Abstract:

A method of enzymatically producing a protein hydrolysate from a protein
substrate is described, wherein a proline-specific endoprotease or a
composition containing a proline-specific endoprotease and optionally a
subtilisin or a metallo endoprotease, and other enzymes such as
carboxypeptidases, is used to produce a protein hydrolysate enriched in
peptide fragments having a carboxy-terminal proline residue. Such protein
hydrolysates may be used as such or to reduce bitterness in foods
nutritionally supplemented by protein hydrolysates, as well as to produce
hydrolysate-containing foodstuffs having low antigenicity.

Claims:

1-21. (canceled)

22. An enzyme composition comprising a proline-specific endoprotease, the
composition being capable of producing a protein hydrolysate comprising
peptides, wherein the molar fraction of peptides (%) carrying a carboxy
terminal proline is at least two times the molar fraction (%) of proline
in the protein

23. The enzyme composition according to claim 22, wherein the composition
further comprises is a serine or a metalloendoprotease or a combination
of serine and metalloendoproteases.

24. (canceled)

25. The enzyme composition according to claim 23, wherein the composition
further comprises at least subtilisin and carboxypeptidase.

26. A method of enzymatically producing a protein hydrolysate from a
protein substrate, which method comprises incubating the protein
substrate with a proline specific endoprotease and one or more other
endoprotease(s), either sequentially or concomitantly, in amounts
sufficient to produce a protein hydrolysate which comprises peptides,
wherein the molar fraction (%) of peptides carrying a carboxy terminal
proline is more than two times the molar fraction (%) of proline in the
protein substrate used to generate the protein hydrolysate.

27. The method according to claim 26, wherein said incubating with the
proline specific endoprotease is performed sequentially to incubating
with the other endoprotease(s).

28. The method according to claim 26, wherein said incubating with the
proline specific endoprotease is performed concomitantly to incubating
with the other endoprotease(s).

29. The method according to claim 26, wherein the proline specific
endoprotease has a pH optimum below 7.

30. The method according to claim 26, wherein the other endoprotease(s) is
a serine or a metalloendoprotease or a combination of serine and
metalloendoproteases.

31. The method according to claim 30, wherein the other endoprotease(s) is
a combination of subtilisin and carboxypeptidase.

32. The method according to claim 26 further comprising recovering the
protein hydrolysate without ultrafiltration or microfiltration.

33. The method according to claim 26, wherein the molar fraction (%) of
peptides carrying a carboxy terminal proline in the protein hydrolysate
is more than three times the molar fraction (%) of proline in the protein
substrate.

34. The method according to claim 26, wherein the proline specific
endoprotease is employed at at least 150 milli-units per gram protein
substrate.

35. The method according to claim 26, wherein the average length of
peptides is from 3 to 9 amino acids.

36. The method according to claim 26, wherein at least 10% of the protein
substrate is hydrolyzed into peptides having molecular masses from 400 to
2000 Dalton.

37. The method according to claim 26, wherein the molar fraction of
peptides carrying a carboxy terminal proline is from 30 to 70%.

38. The method according to claim 26, wherein the protein substrate is
whey and the molar fraction of peptides carrying a carboxy terminal
proline is at least 8%.

39. The method according to claim 26, wherein the protein substrate is
casein and the molar fraction of peptides carrying a carboxy terminal
proline is at least 25%.

40. The method according to claim 26, wherein the protein substrate is soy
and the molar fraction of peptides carrying a carboxy terminal proline is
at least 20%.

41. The method according to claim 26, wherein the protein substrate is
gluten and the molar fraction of peptides carrying a carboxy terminal
proline is at least 20%.

42. The method according to claim 26, wherein the protein substrate is
barley and the molar fraction of peptides carrying a carboxy terminal
proline is at least 20%.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application is a continuation of application Ser. No.
10/433,747, filed Nov. 10, 2003, now allowed; which is a U.S. national
stage filed under 35 U.S.C. 371 of Int'l Appln. No. PCT/EP01/14479; the
entire contents of each of which are hereby incorporated by reference
herein.

FIELD OF THE INVENTION

[0002]The present invention relates to protein hydrolysates, a method to
produce the hydrolysates and the use of these hydrolysates.

BACKGROUND OF THE INVENTION

[0003]Enzyme hydrolysates of cow's milk or fractions of cow's milk have
only limited application in the food industry. Nevertheless, these
hydrolysates occupy interesting niches in the marketplace, as evidenced
by the large volume of literature describing and claiming optimised
processes for obtaining such hydrolysates. Milk or milk fractions are
subjected to enzymes having proteolytic activity to produce the
hydrolysates primarily to minimize the allergenicity of the product,
facilitate gastro-intestinal uptake by offering an easily assimilable
digest, and to stabilize the proteins in acid products against
precipitation during prolonged storage periods.

[0004]Although reducing the molecular weight of milk proteins is commonly
accepted practice for producing these beneficial effects, enzymatic
hydrolysis of milk proteins does have drawbacks. Negative aspects of
incubating milk with enzymes include incomplete proteolytic digestion, an
increasingly bitter taste upon decreasing the length of the peptide
fragments, decreased yields of the final product due to the requisite
purification steps, and unpleasant taste changes caused by high levels of
free amino acids.

[0005]Uniform and complete degradation of all milk fractions via
incubation with endoproteases is often difficult to obtain. For example,
beta-lactoglobulin is known to be protease resistant and partial digests
of this molecule can lead to unexpectedly strong immunogenic reactions to
infant formulae, as well as visible protein precipitations in products
such as acidic sport drinks. To guarantee the absence of inadequately
digested proteins in a protein hydrolysate, a final ultrafiltration step
for the removal of any remaining large peptide fragments from the
hydrolysate is generally required. The indispensable step of removing
these partially digested protein fragments from the hydrolysate
inevitably lowers the yield of the final digestion product, thereby
increasing production costs.

[0006]Protein antigenicity may be overcome by digesting proteins to
peptides having only 8-10 amino acid residues, but the peptides created
by such an extensive proteolytic digestion can be very bitter. The
general explanation for this phenomenon is that smaller peptides with a
high content of hydrophobic amino acids promote bitter tastes. The nature
of the proteinaceous raw material used, the type of proteolytic enzymes
used for digestion and the length of peptides obtained largely determine
the degree of bitterness associated with the final hydrolysate. For
example, casein, which contains many hydrophobic amino acids, is known to
generate far more bitter hydrolysates than whey proteins.

[0007]In industrial operations, debittering of protein hydrolysates is
carried out by the selective removal of bitter peptides using activated
carbon or adsorption to hydrophobic resin. The concomitant yield
reduction during such removal steps increases the cost of the final
product. Moreover, this process has a negative impact on the nutritional
value of the final product, as several nutritionally indispensable amino
acids may be lost due to their hydrophobic nature, including tryptophan,
leucine, phenylalanine and isoleucine. Thus, debittering in this way is
prone to producing hydrolysates deficient in these nutritionally
important amino acids.

[0008]Debittering can also be achieved by subjecting hydrolysates to
exopeptidases. In this approach, amino-terminal and carboxy-terminal
amino acids are liberated from peptides in an attempt to reduce their
overall hydrophobicity. Exposure of peptides to non-selective
exoproteases unfortunately results in the release of uncontrollable
quantities of free amino acids into the final hydrolysate. Subsequent
heating of such hydrolysates containing free amino acids, as required for
sterilisation or spray drying, often generates brothy off-flavors via
Maillard reactions. Moreover, the high levels of free amino acids created
by exoproteases may increase the osmotic value of the final hydrolysate
product to levels that can cause osmotic diarrhoea.

[0009]Therefore, the production of protein hydrolysates represents a
trade-off between the pros and cons of proteolytic digestion. Current
practise is to optimize enzymatic digestion of protein substrates for the
particular requirements of a product category. For example, protein
hydrolysates intended for truly allergic infants require extensive
proteolytic digestion followed by a rigorous removal of any remaining
large molecular weight peptide fragments. By contrast, products designed
for adults, who rarely exhibit bovine milk allergies, typically contain
hydrolysates in which the average peptide length is increased to minimize
the possibility of off flavors and to maximize product yield.

[0010]All major milk proteins, such as beta-casein, beta-lactoglobulin and
alpha-lactoalbumin, as well as vegetable protein fractions obtained from,
for example, soy isolates, rice proteins and wheat gluten are considered
important antigenic compounds. Thus, enzymatic digestion of these milk
and cereal proteins to molecular weights below 3000 Da is considered
important to minimize allergenicity. The beta-lactoglobulin fraction in
whey is especially thought to be an important allergen because this
protein is not present in human milk and proteolytic digestion of
beta-lactoglobulin has proven to be difficult. Infant formula containing
protein hydrolysates that are extensively hydrolyzed typically contain
high levels of free amino acids, which are indicative of suboptimal taste
and high osmolalities. Recent evaluations of currently marketed
hydrolyzed infant formula products have shown that most of them still
contain whey based immunogenic materials. This observation indicates that
new enzyme mixtures leading to improved hydrolysates at a lower cost
continue to be in demand.

[0011]Protein hydrolysates in products destined for consumers with
non-medical needs, for example athletes or people on a slimming diet,
must be tailored to provide good taste characteristics. Under these
circumstances, high palatability as well as physico-chemical aspects,
such as solubility under acidic conditions, are of overriding importance.
Products in this category, including fortified fruit juices and sports
drinks, focus on, inter alia, glutamine and arginine supplementation to
improve consumer health. Sports drinks, for example, serve to enhance
physical endurance and recovery of an athlete after prolonged high
intensity exercise. Glutamine-rich cereal protein sources, like wheat
gluten, or arginine-rich protein sources, like rice protein and soy
isolates, have been considered as alternatives to milk proteins to
satisfy the supplementation needs of acidic health-related products.
However, such cereal proteins, particularly wheat gluten, exhibit very
poor solubilities at more acidic pH values--i.e., those above 4--meaning
completely soluble gluten hydrolysates are difficult to obtain.

[0012]Because of the negative influence on product cost and quality
associated with protein hydrolysis, several enzyme mixtures aimed at
improving hydrolysate characteristics and lowering production costs have
been described in prior publications. Examples include EP 321 603, which
refers to the use of animal-derived endoproteases like trypsin,
chymotrypsin and pancreatin, and EP 325 986 and WO 96/13174, which favor
the use of endoproteases obtained from Bacillus or Aspergillus species.
Several exoproteases have been described as being capable of debittering
mixtures of peptides. Whereas, for example, EP 0223 560 refers to the use
of a specific proline-specific endoprotease, WO 96/13174 refers to a
mixture of amino-peptidases and carboxypeptidases for this purpose.

[0013]A number of publications tout the beneficial effects of
proline-specific endoproteases in combination with various exopeptidases
for producing protein hydrolysates which have relatively low bitterness
profiles. For example, Japanese patent JP 02-039896 refers to the use of
a proline-specific endoprotease combined with a
dipeptidyl-carboxypeptidase for generating low molecular weight peptide
preparations. The degradation of proline-rich oligopeptides by three
proline-specific peptide hydrolases is described as essential for
accelerating cheese ripening without bitterness in the J. Dairy Sci.
77(2): 385-392 (1994). More specifically, the debittering effect of
proline-specific endoprotease in combination with a carboxypeptidase is
described in JP 05-015314. JP 05-015314 describes a crude enzyme
preparation obtained from Aspergillus oryzae that exhibits, apart from a
general, non-specific proteolytic activity, small quantities of a
proline-specific endoprotease and carboxypeptidase activity. According to
JP 05-015314, proline residues present at the carboxy terminii of
peptides cause bitter tastes and are undesirable. Incubation of soy bean
protein with a proline-specific endoprotease and carboxypeptidase enzyme
mixture yielded a hydrolysate that was significantly less bitter than a
soy bean hydolysate obtained with protease preparation lacking the
combination of a proline-specific endoprotease and a carboxypeptidase.

[0014]Collectively, the state of the art strongly suggests that
exopeptidase-mediated release of carboxy-terminal (or amino-terminal)
hydrophobic amino acid residues from peptides is essential for
significantly debittering peptide hydrolysates. Likewise, references that
specifically refer to proline-specific endoproteases for debittering
teach that the function of this activity is to expose the hydrophobic
proline residues to allow their subsequent removal by a carboxypeptidase.
The implication of this hypothesis is that the debittering activity of
proline-specific endoproteases is linked with the efficient removal of
the carboxy-terminal proline residues rather than the creation of
peptides carrying such carboxy-terminal proline residues.

SUMMARY OF THE INVENTION

[0015]The present invention provides a protein hydrolysate which comprises
peptides wherein the molar fraction of peptides (%) carrying a
carboxy-terminal proline is more than two times higher than the molar
fraction (%) of proline in the protein substrate used to generate the
hydrolysate.

The present invention also provides: [0016]a whey hydrolysate which
comprises peptides wherein the molar fraction of peptides carrying a
carboxy-terminal proline is at least 8%, preferably at least 15%, more
preferably from 30 to 70%; [0017]a casein hydrolysate which comprises
peptides wherein the molar fraction of peptide carrying a
carboxy-terminal proline is at least 25%, preferably at least 30% and
more preferably less than 70%; [0018]a soy hydrolysate which comprises
peptides wherein the molar fraction of peptide carrying a
carboxy-terminal proline is at least 20%, preferably from 30 to 70%.
[0019]a gluten hydrolysate which comprises peptides wherein the molar
fraction of peptide carrying a carboxy-terminal proline is at least 20%,
preferably at least 30%, advantageously less than 70%; and [0020]a barley
hydrolysate which comprises peptides wherein the molar fraction of
peptide carrying a carboxy-terminal proline is at least 20%, preferably
at least 30%, advantageously less than 70%.The present invention further
provides a proline-specific endoprotease selected from the group
consisting of:

[0021](a) a polypeptide which has an amino acid sequence which has at
least 40% amino acid sequence identity with amino acids 1 to 526 of SEQ
ID NO: 2 or a fragment thereof;

[0022](b) a polypeptide which is encoded by a polynucleotide which
hybridizes under low stringency conditions with (i) the nucleic acid
sequence of SEQ ID NO: 1 or a fragment thereof which is at least 80% or
90% identical over 60, preferably over 100 nucleotides, more preferably
at least 90% identical over 200 nucleotides, or (ii) a nucleic acid
sequence complementary to the nucleic acid sequence of SEQ ID NO: 1; and

a DNA molecule encoding the endopeptidase.The present invention also
provides:

[0023]the use of a protein hydrolysate of the invention in a food or
drink;

[0024]the use of a proline-specific endoprotease according to the
invention;

[0025]a method of enzymatically producing a protein hydrolysate from a
protein substrate, wherein the protein substrate is incubated with a
proline-specific endoprotease to produce a protein hydrolysate enriched
in peptides having a carboxy-terminal proline;

[0026]an enzyme composition comprising a proline-specific endoprotease of
the invention, the composition being capable of producing a protein
hydrolysate comprising peptides, wherein the molar fraction of peptides
(%) carrying a carboxy-terminal proline is at least two times the molar
fraction (%) of proline in the protein or a hydrolysate of the invention;
and

[0027]a food comprising a protein hydrolysate of the invention or
obtainable by a method of the invention.

[0029]FIG. 2: SDS-PAGE analysis of culture filtrates of the host strain
(A. niger CBS513.88) and several transformants that over-express the
proline-specific endoprotease, here indicated with the arrow.

DETAILED DESCRIPTION OF THE INVENTION

[0030]We have shown that a high incidence of proline residues at the
carboxy-terminal end of peptides can be correlated with low bitterness.
Moreover we have demonstrated that the desired high incidence of
carboxy-terminal proline residues can only be achieved with high
concentrations of a proline-specific endoprotease, i.e., concentrations
that exceed the activity specified in JP 05-015314 by several orders of
magnitude and moreover in the absence of a carboxypeptidase.

[0031]From an economic point of view the implication of this observation
is that there exists a clear need for an improved means of producing
proline-specific endoproteases in high quantities and a relatively pure
form. A preferred way of doing this is via the overproduction of such a
proline-specific endoprotease using recombinant DNA techniques. As many
food products are acidic and long term enzyme incubations under
industrial, non-sterile circumstances require acidic incubation
conditions to prevent microbial contamination, a more preferred way of
doing this is via the overproduction of an acid stable proline-specific
endoprotease using recombinant DNA techniques. A particularly preferred
way of doing this is via the overproduction of an Aspergillus derived
proline-specific endoprotease and a most preferred way of doing this is
via the overproduction of an Aspergillus niger derived proline-specific
endopeptidase. To enable the latter route unique sequence information of
an Aspergillus derived proline-specific endoprotease is essential. More
preferable the whole nucleotide sequence of the encoding gene has to be
available.

[0032]Once the new enzyme has been made available in a relatively pure
form, other new and surprising applications are envisaged which have
technical and economical advantages. A new application would be the
creation of non-bitter hydrolysates from proteinaceous substrates with
unusual amino acid compositions. Such unusual amino acid compositions may
offer serious benefits in certain food applications. Examples are casein
or wheat gluten or maize protein isolate with high levels of hydrophobic
amino acid residues present. Hitherto such substrates were of no
practical use because of the objectional bitter tastes generated upon
hydrolysis using prior art methods. Using the hydrolysis method according
to the invention, new, non-bitter hydrolysates can be made available to
be used in infant and clinical nutrition, in therapeutic diets as well as
in consumer diets and sport nutrition. Apart from such new hydrolysates,
applications that take advantage of the bitterness reducing effect of the
acid proline-specific endoproteases as such are also envisaged. For
example, the incorporation of the endopeptidase in proteinaceous food
products involving a fermentation step such as in cheeses or yogurt to
suppress the bitterness which can evolve upon aging. Also in
proteinaceous food products requiring treatment with proteases such as
the production of enzyme modified cheeses or the production of protein
hydrolysates for the flavor industry, the incorporation of the enzyme
according to the invention will help to suppress bitterness.

[0033]Moreover, benefits not directly related to suppressing bitter tastes
are also investigated.

[0034]One such new application is the incubation of the enzyme with food
proteins to reduce their allergenicity. Several food proteins contain
highly allergenic subfractions, such as wheat gluten that contains
prolamines with proline-rich peptide sequences. These proteins can be
subjected to the new enzyme to alleviate their antigenicity. Another new
application is the incorporation of the enzyme in all kinds of doughs as
it has been observed that this retards the staling of the breads
obtained. Another new application is the use of the proline-specific
endoprotease to generate proline-rich peptides. Such proline-rich
peptides are desirable additions to various food or nutraceutical
products as they have been implicated in anorectic action, in
fibrinolytic and antithrombotic and antihypertensive effects, in
protection of the gastric mucosa as well as the prevention of rheumatoid
arthritis.

[0035]Another surprising application is addition of the new enzyme to
animal feed to enhance protein utilisation. For example, addition of the
enzyme leads to improved digestibility of hard-to-digest proline rich
sequences present in the feed protein as well as to improved conversion
rates of cheaply available vegetable proteins containing high levels of
polyphenols.

[0036]In yet another new application the enzyme is used in beer brewing.
Barley proteins are rich in proline rich sequences and in their
non-malted form cereal proteins are extremely difficult to degrade into
the free amino acids required to create a suitable fermentable wort.
Quite surprisingly the incorporation of the new enzyme into the mashing
process has been shown to stimulate amino acid release from milled but
non-malted barley so that a much richer wort is obtained. In a similar
way beer fermentation from mashes containing a high proportion of other
cheap and locally available cereals such as for example sorghum can be
improved.

[0037]In most of these new applications the proline-specific endoprotease
should preferably exhibit an activity spectrum with an acidic pH optimum.

[0038]To overcome the above-mentioned problems, the invention demonstrates
that the activity of an isolated, purified proline-specific endoprotease
alone--i.e., without the substantial concomitant or subsequent activity
of an exoproteolytic enzyme--is sufficient for significantly debittering
a protein hydrolysate. Therefore the proline-specific endoprotease may
comprise at least 5 units per gram protein of the enzyme preparation of
the invention, preferably 10 U/g, more preferably 25 U/g and even more
preferably 50 U/g. Moreover, studies conducted in accordance with the
invention demonstrate that the activity of an isolated, purified
proline-specific endoprotease alone, meaning without the concomitant or
subsequent activity of an exoproteolytic enzyme, is sufficient to
significantly decrease the overall immunogenicity level of protein
hydrolysates, as well as to significantly increase their overall
solubility under acidic conditions. The hydrolysates produced according
to the invention are enriched in peptides having a carboxy-terminal
proline residue.

[0039]An embodiment of the present invention provides an enzyme mixture
comprising an isolated, purified proline-specific endoprotease for the
high yield production of protein hydrolysates having substantially low
bitterness and low allergenic properties without the concomitant
production of substantial levels of free amino acids. This enzyme mixture
is suitable for preparing hydrolysates of various protein fractions. In
particular, a protein substrate, such as a milk protein, may be incubated
with an isolated, purified proline-specific endoprotease and a subtilisin
to produce a protein hydrolysate enriched in peptide fragments having a
carboxy-terminal proline. The term "enriched" is intended to mean that at
least 8% of the peptide fragments in the hydrolysate product of enzymatic
cleavage possess a carboxy-terminal proline residue.

[0040]The present invention provides a protein hydrolysate obtained by
hydrolysing a protein which comprises peptides wherein the molar fraction
of peptides (%) carrying a carboxy-terminal proline is at least two times
the molar fraction (%) of proline in the protein substrate used to
produce the hydrolysate.

[0041]The average length of the peptides in the hydrolysates is in general
from 3 to 9 amino acids.

[0042]Preferred hydrolysates according to the invention are: a whey
hydrolysate which comprises peptides wherein the molar fraction of
peptides carrying a carboxy-terminal proline is at least 8%, preferably
at least 15%, more preferably from 30 to 70%, a casein hydrolysate which
comprises peptides wherein the molar fraction of peptide carrying a
carboxy-terminal proline is at least 25%, preferably from 30 to 70%, and
a soy hydrolysate which comprises peptides wherein the molar fraction of
peptides carrying a carboxy-terminal proline is at least 20%, preferably
from 30 to 70%.

[0043]By peptides or peptide fragments it is meant peptides with molecular
masses from 400 to 2000 Dalton. These peptides can be analyzed according
to the LC/MC analysis as described the Materials & Methods section.

[0044]In general in the production of the protein hydrolysates of the
invention the protein substrate is substantially hydrolyzed,
advantageously for at least 50%. Preferably at least 10% of the protein
substrate is converted into peptides having molecular masses from 400 to
2000 Dalton. More preferably from 20 to 90% and even more preferably from
30 to 80% of the protein substrate is converted into such peptides.

[0045]In another embodiment of the invention, a protein substrate may be
incubated with an enzyme mixture comprising an isolated, purified
proline-specific endoprotease, a serine endoprotease or a metallo
endoprotease and a carboxypeptidase to produce a protein hydrolysate
enriched in peptide fragments having a carboxy-terminal proline.

[0046]The enzyme mixture of the invention is particularly suitable for use
in the production of protein hydrolysates intended for flavoring and
nutrient enhancement of sport drinks and juice-based beverages, as the
resulting hydrolyzed peptide mixture combines a very low bitterness
profile with excellent solubility under the prevailing acidic conditions
of such beverages. The enzyme mixture of the invention is characterized
in that it contains at least one endoprotease for example a serine
protease or a metallo endoprotease in conjunction with a proline-specific
endoprotease (E.C. 3.4.21.26) to provide a primary hydrolysate. More
specifically, the invention relates to an isolated, purified
proline-specific endoprotease and a serine protease or metallo protease
enzyme mixture capable of producing a protein hydrolysate comprising
peptide fragments, wherein at least 8%, preferably at least 15%, more
preferably from 30 to 70% of said peptide fragments have a
carboxy-terminal proline.

[0047]Another embodiment of the invention is a protein hydrolysate
enriched with a relatively high content of peptides having proline as the
carboxy-terminal amino acid residue. Such enriched hydrolysates may
comprise at least 8%, preferably at least 15%, more preferably from 30 to
70% peptide fragments having a carboxy-terminal proline residue. Since
enzyme preparations typically utilized in the genesis of protein
hydrolysates are not capable of generating peptides bearing proline
residues at carboxy-terminii, protein hydrolysates that are relatively
rich in such peptides are novel.

[0048]Substrates for hydrolysis by an enzyme mixture of the invention
include whole milk, skimmed milk, acid casein, rennet casein, acid whey
products or cheese whey products. Quite surprisingly the Aspergillus
derived proline-specific endoprotease doesnot only cleave at the
carboxy-terminal side of proline residues but also at the
carboxy-terminal side of hydroxyproline residues which makes other,
collagen based animal proteins such as gelatine as well as bones or
fish-bones containing residual meat, interesting substrates for the
enzyme. Moreover, vegetable substrates like wheat gluten, milled barley
and protein fractions obtained from, for example, soy, rice or corn are
suitable substrates. Milk protein hydrolysates produced according to the
invention may be used with or without additional filtration or
purification steps in various speciality foods such as hypoallergenic
hydrolysates for infant nutrition, basic hydrolysates for enteral and
dietetic nutrition, as well as protein concentrates for various forms of
health food. Thus, protein hydrolysates of the invention may be used to
produce foodstuffs having low antigenicity, such as infant formula. In
addition, enzyme preparations according to the invention may be used to
reduce bitterness in foods flavored by at least one protein hydrolysate,
even when the protein hydrolysate is present in large amounts. For
example, foods may comprise between 5% and 10% (w/v) of a protein
hydrolysate and still have their bitterness reduced using an enzyme
preparation of the invention.

[0049]The present invention provides an isolated, purified
proline-specific endoprotease with an acidic pH optimum alone or in a
composition comprising one or more additional enzymes for the preparation
of a protein hydrolysate for various food applications. Such an isolated,
purified proline-specific endoprotease is defined to have at least 10
units of proline-specific endoprotease activity per gram of proteinaceous
material. These units should be measured using the synthetic peptide
Z-Gly-Pro-pNA at 37° C. and pH 5 in case the pH optimum of the
proline-specific endoprotease is below pH 6, for example in case of
Aspergillus niger proline-specific endo protease or else the units should
be measured at pH=7, as specified in the Materials & Methods section.
This isolated, purified enzyme, alone or in an enzyme mixture, overcomes
a number of disadvantages of enzyme mixtures previously known in the art.
Most importantly, the inventive isolated, purified proline-specific
endoprotease is key in the production of hydrolysates which combine a low
allergenic potential, a high yield and a low bitterness profile.
Moreover, the hydrolysates produced with the isolated, purified
proline-specific endoprotease or an enzyme mixture comprising this
proline-specific endoprotease are acid stable and contain very low levels
of free amino acids, such that minimal off-tastes are generated during
heating steps, such as spray drying or product sterilisation.
Hydrolysates according to the invention will contain less than 900
micromoles of free amino acids per gram of dry powder, preferably less
than 300 micromoles of free amino acids per gram of dry powder, more
preferably less than 150 micromoles of free amino acids per gram of dry
powder, and even more preferably less than 50 micromoles per gram of dry
powder.

[0050]The enzyme mixture according to the invention is characterized in
that it comprises another endoprotease such as a serine protease or a
metallo endoprotease in conjunction with an isolated, purified
proline-specific endoprotease (E.C. 3.4.21.26) which work together to
provide a primary protein hydrolysate.

[0051]Serine proteases represent a well-known class of alkaline
endoproteases and some of its most important representants such as
subtilisin (E.C. 3.4.21.62) and chymotrypsin (E.C. 3.4.21.1) prefer
cleavage of the peptide chain at the carboxy-terminal side of hydrophobic
amino acids such as Tyr, Trp, Phe and Leu. The enzyme mixture of the
invention may contain chymotrypsin and/or subtilisin. Subtilisin is
produced by species of Bacillus, has a particularly broad substrate
specificity and a broad, alkaline pH optimum. The enzyme is optimally
active between 50° C. and 60° C. The enzyme is cheaply
available as a regular commercial product and is useful in the production
of, for example, various milk hydrolysates. Chymotrypsin may be obtained
from animal pancreases, has a somewhat narrower substrate specificity at
slightly more alkaline pH values than subtilisin and is optimally active
below 50° C.

[0052]The class of metallo-endoproteases is wide spread in bacteria, fungi
and higher organisms. They can be separated into the neutral and acid
metalloproteases. Of these two subclasses only the neutral proteases
exhibit the desirable cleavage preference, i.e., cleaving the peptide
chain on the carboxy-terminal side of hydrophobic amino acid residues
such as Phe and Leu. Well-known representatives of the category of the
neutral metalloproteases are bacillolysin (E.C. 3.4.24.28) and
thermolysin (E.C. 3.4.24.27) and either, or both of these, may be present
in the enzyme mixture of the invention. Both enzymes are obtained from
Bacillus species and exhibit maximum activity under neutral or slightly
alkaline conditions. Less well-known representatives of these neutral
metalloendoproteases have been obtained from Aspergillus species. In
those cases in which the proline-specific endoprotease is not used for
its debittering effects but to aid in the hydrolysis of proline rich
protein sequences, combinations with the class of the acid
metalloproteases, as for example deuterolysine (EC 3.4.24.39) can be
advantageous.

[0053]A proline-specific endoprotease is an endoprotease capable of
cleaving peptides or polypeptides at the carboxy-terminal end of proline
residues. Such enzymes are widely found in animals and plants, but their
presence in microorganisms appears to be limited. To date,
proline-specific endoprotease have been identified in species of
Aspergillus (EP 0 522 428), Flavobacterium (EP 0 967 285) and Aeromonas
(J. Biochem. 113: 790-796), Xanthomonas and Bacteroides. Though the
proline-specific enzymes from most of these organisms are active around
pH 8, the Aspergillus enzyme is optimally active around pH 5. According
to a preferred embodiment, proline-specific endoprotease having a pH
optimum below 7, preferably having a pH optimum from 3.5 to 6.5 is used
because of the technical and economical advantages of such enzymes. The
proline-specific endoprotease of the invention may be isolated from one
of the above-mentioned microbial species, particularly from a species of
Aspergillus. Preferably, the proline-specific endoprotease is isolated
from a strain of Aspergillus niger. More preferably, the proline-specific
endoprotease is isolated from an Aspergillus niger host engineered to
overexpress a gene encoding a proline-specific endoprotease, although
other hosts, such as E. coli are suitable expression vectors. For
example, the cloning and overproduction of the Flavobacterium derived
proline-specific endoprotease in, among others, E. coli has made certain
proline-specific endoproteases available in a pure form. An example of
such an overproducing construct is provided in the World Journal of
Microbiology & Biotechnology, 11, 209-212 (1995). An Aspergillus niger
host is preferably used to produce a non-recombinant self-construct
utilizing A. niger promoters to drive the expression of a gene encoding
an A. niger proline-specific endoprotease.

[0054]Most of the scientific publications concerning the cloning and
production of proline-specific-endoproteases focus on the role of this
enzyme in the synthesis and regulation of biologically active proteins.
Publications implicating this enzyme in the production of useful protein
hydrolysates are scarce and are concerned with the use of the enzyme in
conjunction with an exoprotease. Several Japanese publications refer to
the presence of proline-specific-endoproteolytic activity in crude and
complex enzyme mixtures capable of producing hydrolysates with low
bitterness profiles, but the enzyme mixtures used always contain
exoproteases. No direct connection between debittering and
proline-specific endoproteolytic activity in the absence of exoproteases
like carboxypeptidases or aminopeptidases is suggested in the art.
Moreover, no data linking hydrolysates produced using
proline-specific-endoproteolytic activity with a diminished immunogenic
response or an improved acid solubility has been previously described.

[0055]A polypeptide of the invention which has proline-specific
endoprotease may be in an isolated form. As defined herein, an isolated
polypeptide is an endogenously produced or a recombinant polypeptide
which is essentially free from other non-proline-specific endoprotease
polypeptides, and is typically at least about 20% pure, preferably at
least about 40% pure, more preferably at least about 60% pure, even more
preferably at least about 80% pure, still more preferably about 90% pure,
and most preferably about 95% pure, as determined by SDS-PAGE. The
polypeptide may be isolated by centrifugation and chromatographic
methods, or any other technique known in the art for obtaining pure
proteins from crude solutions. It will be understood that the polypeptide
may be mixed with carriers or diluents which do not interfere with the
intended purpose of the polypeptide, and thus the polypeptide in this
form will still be regarded as isolated. It will generally comprise the
polypeptide in a preparation in which more than 20%, for example more
than 30%, 40%, 50%, 80%, 90%, 95% or 99%, by weight of the proteins in
the preparation is a polypeptide of the invention.

[0056]Preferably, the polypeptide of the invention is obtainable from a
microorganism which possesses a gene encoding an enzyme with
proline-specific endoprotease activity. More preferably the microorganism
is fungal, and optimally is a filamentous fungus. Preferred organisms are
thus of the genus Aspergillus, such as those of the species Aspergillus
niger.

[0057]In a first embodiment, the present invention provides an isolated
polypeptide having an amino acid sequence which has a degree of amino
acid sequence identity to amino acids 1 to 526 of SEQ ID NO: 2 (i.e., the
polypeptide) of at least about 40%, preferably at least about 50%,
preferably at least about 60%, preferably at least about 65%, preferably
at least about 70%, more preferably at least about 80%, even more
preferably at least about 90%, still more preferably at least about 95%,
and most preferably at least about 97%, and which has proline-specific
endoprotease activity.

[0058]For the purposes of the present invention, the degree of identity
between two or more amino acid sequences is determined by BLAST P protein
database search program (Altschul et al., Nucl. Acids Res. 25: 3389-3402,
1997) with matrix Blosum 62 and an expected threshold of 10.

[0059]A polypeptide of the invention may comprise the amino acid sequence
set forth in SEQ ID NO: 2 or a substantially homologous sequence, or a
fragment of either sequence having proline-specific endoprotease
activity. In general, the naturally occurring amino acid sequence shown
in SEQ ID NO: 2 is preferred.

[0060]The polypeptide of the invention may also comprise a naturally
occurring variant or species homologue of the polypeptide of SEQ ID NO:
2.

[0061]A variant is a polypeptide that occurs naturally in, for example,
fungal, bacterial, yeast or plant cells, the variant having
proline-specific endoprotease activity and a sequence substantially
similar to the protein of SEQ ID NO: 2. The term "variants" refers to
polypeptides which have the same essential character or basic biological
functionality as the proline-specific endoprotease of SEQ ID NO: 2, and
includes allelic variants. The essential character of proline-specific
endoprotease of SEQ ID NO: 2 is that it is an enzyme capable of cleaving
the amino-terminal amino acid from a protein or (poly)peptide.
Preferably, a variant polypeptide has at least the same level of
proline-specific endoprotease activity as the polypeptide of SEQ ID NO:
2. Variants include allelic variants either from the same strain as the
polypeptide of SEQ ID NO: 2. or from a different strain of the same genus
or species.

[0062]Similarly, a species homologue of the inventive protein is an
equivalent protein of similar sequence which is a proline-specific
endoprotease and occurs naturally in another species of Aspergillus.

[0063]Variants and species homologues can be isolated using the procedures
described herein which were used to isolate the polypeptide of SEQ ID NO:
2 and performing such procedures on a suitable cell source, for example a
bacterial, yeast, fungal or plant cell. Also possible is to use a probe
of the invention to probe libraries made from yeast, bacterial, fungal or
plant cells in order to obtain clones expressing variants or species
homologues of the polypepetide of SEQ ID NO: 2. These clones can be
manipulated by conventional techniques to generate a polypeptide of the
invention which thereafter may be produced by recombinant or synthetic
techniques known per se.

[0064]The sequence of the polypeptide of SEQ ID NO: 2 and of variants and
species homologues can also be modified to provide polypeptides of the
invention. Amino acid substitutions may be made, for example from 1, 2 or
3 to 10, 20 or 30 substitutions. The same number of deletions and
insertions may also be made. These changes may be made outside regions
critical to the function of the polypeptide, as such a modified
polypeptide will retain its proline-specific endoprotease activity.

[0065]Polypeptides of the invention include fragments of the above
mentioned full length polypeptides and of variants thereof, including
fragments of the sequence set out in SEQ ID NO: 2. Such fragments will
typically retain activity as a proline-specific endoprotease. Fragments
may be at least 50, 100 or 200 amino acids long or may be this number of
amino acids short of the full length sequence shown in SEQ ID NO: 2.

[0066]Polypeptides of the invention can, if necessary, be produced by
synthetic means although usually they will be made recombinantly as
described below. Synthetic polypeptides may be modified, for example, by
the addition of histidine residues or a T7 tag to assist their
identification or purification, or by the addition of a signal sequence
to promote their secretion from a cell.

[0067]Thus, the variants sequences may comprise those derived from strains
of Aspergillus other than the strain from which the polypeptide of SEQ ID
NO: 2 was isolated. Variants can be identified from other Aspergillus
strains by looking for proline-specific endoprotease activity and cloning
and sequencing as described herein. Variants may include the deletion,
modification or addition of single amino acids or groups of amino acids
within the protein sequence, as long as the peptide maintains the basic
biological functionality of the proline-specific endoprotease of SEQ ID
NO: 2.

[0068]Amino acid substitutions may be made, for example from 1, 2 or from
3 to 10, 20 or 30 substitutions. The modified polypeptide will generally
retain activity as a proline-specific endoprotease. Conservative
substitutions may be made; such substitutions are well-known in the art.
Preferably substitutions do not affect the folding or activity of the
polypeptide.

[0069]Shorter polypeptide sequences are within the scope of the invention.
For example, a peptide of at least 50 amino acids or up to 60, 70, 80,
100, 150 or 200 amino acids in length is considered to fall within the
scope of the invention as long as it demonstrates the basic biological
functionality of the proline-specific endoprotease of SEQ ID NO: 2. In
particular, but not exclusively, this aspect of the invention encompasses
the situation in which the protein is a fragment of the complete protein
sequence.

[0070]In a second embodiment, the present invention provides an to
isolated polypeptide which has proline-specific endoprotease activity,
and is encoded by polynucleotides which hybridize or are capable of
hybrizing under low stringency conditions, more preferably medium
stringency conditions, and most preferably high stringency conditions,
with (I) the nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid
fragment comprising at least the C-terminal portion of SEQ ID NO: 1, but
having less than all or having bases differing from the bases of SEQ ID
NO: 1; or (ii) with a nucleic acid strand complementary to SEQ ID NO: 1.

[0071]The term "capable of hybridizing" means that the target
polynucleotide of the invention can hybridize to the nucleic acid used as
a probe (for example, the nucleotide sequence set forth in SEQ. ID NO: 1,
or a fragment thereof, or the complement of SEQ ID NO: 1) at a level
significantly above background. The invention also includes the
polynucleotides that encode the proline-specific endoprotease of the
invention, as well as nucleotide sequences which are complementary
thereto. The nucleotide sequence may be RNA or DNA, including genomic
DNA, synthetic DNA or cDNA. Preferably, the nucleotide sequence is DNA
and most preferably, a genomic DNA sequence. Typically, a polynucleotide
of the invention comprises a contiguous sequence of nucleotides which is
capable of hybridizing under selective conditions to the coding sequence
or the complement of the coding sequence of SEQ ID NO: 1. Such
nucleotides can be synthesized according to methods well-known in the
art.

[0072]A polynucleotide of the invention can hybridize to the coding
sequence or the complement of the coding sequence of SEQ ID NO: 1 at a
level significantly above background. Background hybridization may occur,
for example, because of other cDNAs present in a cDNA library. The signal
level generated by the interaction between a polynucleotide of the
invention and the coding sequence or complement of the coding sequence of
SEQ ID NO: 1 is typically at least 10 fold, preferably at least 20 fold,
more preferably at least 50 fold, and even more preferably at least 100
fold, as intense as interactions between other polynucleotides and the
coding sequence of SEQ ID NO: 1. The intensity of interaction may be
measured, for example, by radiolabelling the probe, for example with
32P. Selective hybridization may typically be achieved using
conditions of low stringency (0.3M sodium chloride and 0.03M sodium
citrate at about 40° C.), medium stringency (for example, 0.3M
sodium chloride and 0.03M sodium citrate at about 50° C.) or high
stringency (for example, 0.3M sodium chloride and 0.03M sodium citrate at
about 60° C.).

Modifications

[0073]Polynucleotides of the invention may comprise DNA or RNA. They may
be single or double stranded. They may also be polynucleotides which
include within them synthetic or modified nucleotides including peptide
nucleic acids. A number of different types of modifications to
polynucleotides are known in the art. These include a methylphosphonate
and phosphorothioate backbones, and addition of acridine or polylysine
chains at the 3' and/or 5' ends of the molecule. For the purposes of the
present invention, it is to be understood that the polynucleotides
described herein may be modified by any method available in the art.

[0074]It is to be understood that skilled persons may, using routine
techniques, make nucleotide substitutions that do not affect the
polypeptide sequence encoded by the polynucleotides of the invention to
reflect the codon usage of any particular host organism in which the
polypeptides of the invention are to be expressed.

[0075]The coding sequence of SEQ ID NO: 1 may be modified by nucleotide
substitutions, for example from 1, 2 or 3 to 10, 25, 50 or 100
substitutions. The polynucleotide of SEQ ID NO: 1 may alternatively or
additionally be modified by one or more insertions and/or deletions
and/or by an extension at either or both ends. The modified
polynucleotide generally encodes a polypeptide which has proline-specific
endoprotease activity. Degenerate substitutions may be made and/or
substitutions may be made which would result in a conservative amino acid
substitutions when the modified sequence is translated, for example as
discussed with reference to polypeptides later.

Homologues

[0076]A nucleotide sequence which is capable of selectively hybridizing to
the complement of the DNA coding sequence of SEQ ID NO: 1 is included in
the invention and will generally have at least 50% or 60%, at least 70%,
at least 80%, at least 90%, at least 95%, at least 98% or at least 99%
sequence identity to the coding sequence of SEQ ID NO: 1 over a region of
at least 60, preferably at least 100, more preferably at least 200
contiguous nucleotides or most preferably over the full length of SEQ ID
NO: 1. Likewise, a nucleotide which encodes an active proline-specific
endoprotease and which is capable of selectively hybridizing to a
fragment of a complement of the DNA coding sequence of SEQ ID NO: 1, is
also embraced by the invention. A C-terminal fragment of the nucleic acid
sequence of SEQ ID NO: 1 which is at least 80% or 90% identical over 60,
preferably over 100 nucleotides, more preferably at least 90% identical
over 200 nucleotides is encompassed by the invention.

[0077]Any combination of the above mentioned degrees of identity and
minimum sizes may be used to define polynucleotides of the invention,
with the more stringent combinations (i.e., higher identity over longer
lengths) being preferred. Thus, for example, a polynucleotide which is at
least 80% or 90% identical over 60, preferably over 100 nucleotides,
forms one aspect of the invention, as does a polynucleotide which is at
least 90% identical over 200 nucleotides.

[0078]The UWGCG Package provides the BESTFIT program which may be used to
calculate identity (for example used on its default settings).

[0079]The PILEUP and BLAST N algorithms can also be used to calculate
sequence identity or to line up sequences (such as identifying equivalent
or corresponding sequences, for example on their default settings).

[0080]Software for performing BLAST analyses is publicly available through
the National Center for Biotechnology Information
(www-dot-ncbi-dot-nlm-dot-nih-dot-gov). This algorithm involves first
identifying high scoring sequence pair (HSPs) by identifying short words
of length W in the query sequence that either match or satisfy some
positive-valued threshold score T when aligned with a word of the same
length in a database sequence. T is referred to as the neighborhood word
score threshold. These initial neighborhood word hits act as seeds for
initiating searches to find HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Extensions for the word hits
in each direction are halted when: the cumulative alignment score falls
off by the quantity X from its maximum achieved value; the cumulative
score goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence is
reached. The BLAST algorithm parameters W, T and X determine the
sensitivity and speed of the alignment. The BLAST program uses as
defaults a word length (W) of 11, the BLOSUM62 scoring matrix alignments
(B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both
strands.

[0081]The BLAST algorithm performs a statistical analysis of the
similarity between two sequences. One measure of similarity provided by
the BLAST algorithm is the smallest sum probability (P(N)), which
provides an indication of the probability by which a match between two
nucleotide or amino acid sequences would occur by chance. For example, a
sequence is considered similar to another sequence if the smallest sum
probability in comparison of the first sequence to the second sequence is
less than about 1, preferably less than about 0.1, more preferably less
than about 0.01, and most preferably less than about 0.001.

Primers and Probes

[0082]Polynucleotides of the invention include and may be used as primers,
for example as polymerase chain reaction (PCR) primers, as primers for
alternative amplification reactions, or as probes for example labelled
with a revealing label by conventional means using radioactive or
non-radioactive labels, or the polynucleotides may be cloned into
vectors. Such primers, probes and other fragments will be at least 15,
for example at least 20, 25, 30 or 40 nucleotides in length. They will
typically be up to 40, 50, 60, 70, 100, 150, 200 or 300 nucleotides in
length, or even up to a few nucleotides (such as 5 or 10 nucleotides)
short of the coding sequence of SEQ ID NO: 1.

[0083]In general, primers will be produced by synthetic means, involving a
step-wise manufacture of the desired nucleic acid sequence one nucleotide
at a time. Techniques for accomplishing this using automated protocols
are readily available in the art. Longer polynucleotides will generally
be produced using recombinant means, for example using PCR cloning
techniques. This will involve making a pair of primers (typically of
about 15-30 nucleotides) to amplify the desired region of the
proline-specific endoprotease to be cloned, bringing the primers into
contact with mRNA, cDNA or genomic DNA obtained from a yeast, bacterial,
plant, prokaryotic or fungal cell, preferably of an Aspergillus strain,
performing a polymerase chain reaction under conditions suitable for the
amplification of the desired region, isolating the amplified fragment
(e.g., by purifying the reaction mixture on an agarose gel) and
recovering the amplified DNA. The primers may be designed to contain
suitable restriction enzyme recognition sites so that the amplified DNA
can be cloned into a suitable cloning vector.

[0084]Such techniques may be used to obtain all or part of the
polynucleotides encoding the proline-specific endoprotease sequences
described herein. Introns, promoter and trailer regions are within the
scope of the invention and may also be obtained in an analogous manner
(e.g., by recombinant means, PCR or cloning techniques), starting with
genomic DNA from a fungal, yeast, bacterial plant or prokaryotic cell.

[0085]The polynucleotides or primers may carry a revealing label. Suitable
labels include radioisotopes such as 32P or 35S, enzyme labels,
or other protein labels such as biotin. Such labels may be added to
polynucleotides or primers of the invention and may be detected using
techniques known to persons skilled in the art.

[0086]Polynucleotides or primers (or fragments thereof) labelled or
unlabelled may be used in nucleic acid-based tests for detecting or
sequencing a proline-specific endoprotease or a variant thereof in a
fungal sample. Such detection tests will generally comprise bringing a
fungal sample suspected of containing the DNA of interest into contact
with a probe comprising a polynucleotide or primer of the invention under
hybridizing conditions, and detecting any duplex formed between the probe
and nucleic acid in the sample. Detection may be achieved using
techniques such as PCR or by immobilizing the probe on a solid support,
removing any nucleic acid in the sample which is not hybridized to the
probe, and then detecting any nucleic acid which is hybridized to the
probe. Alternatively, the sample nucleic acid may be immobilized on a
solid support, the probe hybridized and the amount of probe bound to such
a support after the removal of any unbound probe detected.

[0087]The probes of the invention may conveniently be packaged in the form
of a test kit in a suitable container. In such kits the probe may be
bound to a solid support where the assay format for which the kit is
designed requires such binding. The kit may also contain suitable
reagents for treating the sample to be probed, hybridizing the probe to
nucleic acid in the sample, control reagents, instructions, and the like.
The probes and polynucleotides of the invention may also be used in
microassay.

[0088]Preferably; the polynucleotide of the invention is obtainable from
the same organism as the polypeptide, such as a fungus, in particular a
fungus of the genus Aspergillus.

[0089]The polynucleotides of the invention also include variants of the
sequence of SEQ ID NO: 1 which encode for a polypeptide having
proline-specific endoprotease activity. Variants may be formed by
additions, substitutions and/or deletions. Such variants of the coding
sequence of SEQ ID NO: 1 may thus encode polypeptides which have the
ability to digest a polypeptide chain at the carboxy-terminal side of
proline.

Production of Polynucleotides

[0090]Polynucleotides which do not have 100% identity with SEQ ID NO: 1
but fall within the scope of the invention can be obtained in a number of
ways. Thus, variants of the proline-specific endoprotease sequence
described herein may be obtained for example, by probing genomic DNA
libraries made from a range of organisms, such as those discussed as
sources of the polypeptides of the invention. In addition, other fungal,
plant or prokaryotic homologues of proline-specific endoprotease may be
obtained and such homologues and fragments thereof in general will be
capable of hybridizing to SEQ ID NO: 1. Such sequences may be obtained by
probing cDNA libraries or genomic DNA libraries from other species, and
probing such libraries with probes comprising all or part of SEQ ID. 1
under conditions of low, medium to high stringency (as described
earlier). Nucleic acid probes comprising all or part of SEQ ID NO: 1 may
be used to probe cDNA or genomic libraries from other species, such as
those described as sources for the polypeptides of the invention.

[0091]Species homologues may also be obtained using degenerate PCR, which
uses primers designed to target sequences within the variants and
homologues which encode conserved amino acid sequences. The primers can
contain one or more degenerate positions and will be used at stringency
conditions lower than those used for cloning sequences with single
sequence primers against known sequences.

[0092]Alternatively, such polynucleotides may be obtained by site directed
mutagenesis of the proline-specific endoprotease sequences or variants
thereof. This may be useful where, for example, silent codon changes to
sequences are required to optimize codon preferences for a particular
host cell in which the polynucleotide sequences are being expressed.
Other sequence changes may be made in order to introduce restriction
enzyme recognition sites, or to alter the property or function of the
polypeptides encoded by the polynucleotides.

[0093]The invention includes double stranded polynucleotides comprising a
polynucleotide of the invention and its complement.

[0094]The present invention also provides polynucleotides encoding the
polypeptides of the invention described above. Since such polynucleotides
will be useful as sequences for recombinant production of polypeptides of
the invention, it is not necessary for them to be capable of hybridizing
to the sequence of SEQ ID NO: 1, although this will generally be
desirable. Otherwise, such polynucleotides may be labelled, used, and
made as described above if desired.

Recombinant Polynucleotides

[0095]The invention also provides vectors comprising a polynucleotide of
the invention, including cloning and expression vectors, and in another
aspect methods of growing, transforming or transfecting such vectors into
a suitable host cell, for example under conditions in which expression of
a polypeptide of, or encoded by a sequence of, the invention occurs.
Provided also are host cells comprising a polynucleotide or vector of the
invention wherein the polynucleotide is heterologous to the genome of the
host cell. The term "heterologous", usually with respect to the host
cell, means that the polynucleotide does not naturally occur in the
genome of the host cell or that the polypeptide is not naturally produced
by that cell. Preferably, the host cell is a yeast cell, for example a
yeast cell of the genus Kluyveromyces or Saccharomyces or a filamentous
fungal cell, for example of the genus Aspergillus.

[0096]Polynucleotides of the invention can be incorporated into a
recombinant replicable vector, for example a cloning or expression
vector. The vector may be used to replicate the nucleic acid in a
compatible host cell. Thus, in a further embodiment, the invention
provides a method of making polynucleotides of the invention by
introducing a polynucleotide of the invention into a replicable vector,
introducing the vector into a compatible host cell, and growing the host
cell under conditions which bring about replication of the vector. The
vector may be recovered from the host cell. Suitable host cells are
described below in connection with expression vectors.

Vectors

[0097]The vector into which the expression cassette of the invention is
inserted may be any vector that may conveniently be subjected to
recombinant DNA procedures, and the choice of the vector will often
depend on the host cell into which it is to be introduced. Thus, the
vector may be an autonomously replicating vector--i.e., a vector which
exists as an extra-chromosomal entity--the replication of which is
independent of chromosomal replication, such as a plasmid. Alternatively,
the vector may be one which, when introduced into a host cell, is
integrated into the host cell genome and replicates together with the
chromosome(s) into which it has been integrated.

[0098]Preferably, when a polynucleotide of the invention is in a vector it
is operably linked to a regulatory sequence which is capable of providing
for the expression of the coding sequence by the host cell, i.e., the
vector is an expression vector. The term "operably linked" refers to a
juxtaposition wherein the components described are in a relationship
permitting them to function in their intended manner. A regulatory
sequence such as a promoter, enhancer or other expression regulation
signal "operably linked" to a coding sequence is positioned in such a way
that expression of the coding sequence is achieved under conditions
compatible with the control sequences.

[0099]The vectors may, for example in the case of plasmid, cosmid, virus
or phage vectors, be provided with an origin of replication, optionally a
promoter for the expression of the polynucleotide and optionally an
enhancer and/or a regulator of the promoter. A terminator sequence may be
present, as may be a polyadenylation sequence. The vectors may contain
one or more selectable marker genes, for example an ampicillin resistance
gene in the case of a bacterial plasmid or a neomycin resistance gene for
a mammalian vector. Vectors may be used in vitro, for example for the
production of RNA or can be used to transfect or transform a host cell.

[0100]The DNA sequence encoding the polypeptide is preferably introduced
into a suitable host as part of an expression construct in which the DNA
sequence is operably linked to expression signals which are capable of
directing expression of the DNA sequence in the host cells. For
transformation of the suitable host with the expression construct
transformation procedures are available which are well-known to the
skilled person. The expression construct can be used for transformation
of the host as part of a vector carrying a selectable marker, or the
expression construct is co-transformed as a separate molecule together
with the vector carrying a selectable marker. The vectors may contain one
or more selectable marker genes.

[0101]Preferred selectable markers include but are not limited to those
that complement a defect in the host cell or confer resistance to a drug.
They include for example versatile marker genes that can be used for
transformation of most filamentous fungi and yeasts such as acetamidase
genes or cDNAs (the amdS, niaD, facA genes or cDNAs from A. nidulans, A.
oryzae, or A. niger), or genes providing resistance to antibiotics like
G418, hygromycin, bleomycin, kanamycin, phleomycin or benomyl resistance
(benA). Alternatively, specific selection markers can be used such as
auxotrophic markers which require corresponding mutant host strains:
e.g., URA3 (from S. cerevisiae or analogous genes from other yeasts),
pyrG or pyrA (from A. nidulans or A. niger), argB (from A. nidulans or A.
niger) or trpC. In a preferred embodiment the selection marker is deleted
from the transformed host cell after introduction of the expression
construct so as to obtain transformed host cells capable of producing the
polypeptide which are free of selection marker genes.

[0102]Other markers include ATP synthetase subunit 9 (oliC),
orotidine-5'-phosphate-decarboxylase (pvrA), the bacterial G418
resistance gene (useful in yeast, but not in filamentous fungi), the
ampicillin resistance gene (E. coli), the neomycin resistance gene
(Bacillus) and the E. coli uidA gene, coding for glucuronidase (GUS).
Vectors may be used in vitro, for example for the production of RNA or to
transfect or transform a host cell.

[0103]For most filamentous fungi and yeast, the expression construct is
preferably integrated into the genome of the host cell in order to obtain
stable transformants. However, for certain yeasts suitable episomal
vector systems are also available into which the expression construct can
be incorporated for stable and high level expression. Examples thereof
include vectors derived from the 2 μm, CEN and pKD1 plasmids of
Saccharomyces and Kluyveromyces, respectively, or vectors containing an
AMA sequence (e.g., AMA1 from Aspergillus). When expression constructs
are integrated into host cell genomes, the constructs are either
integrated at random loci in the genome, or at predetermined target loci
using homologous recombination, in which case the target loci preferably
comprise a highly expressed gene. A highly expressed gene is a gene whose
mRNA can make up at least 0.01% (w/w) of the total cellular mRNA, for
example under induced conditions, or alternatively, a gene whose gene
product can make up at least 0.2% (w/w) of the total cellular protein,
or, in case of a secreted gene product, can be secreted to a level of at
least 0.05 g/I.

[0104]An expression construct for a given host cell will usually contain
the following elements operably linked to each other in consecutive order
from the 5'-end to 3'-end relative to the coding strand of the sequence
encoding the polypeptide of the first aspect: (1) a promoter sequence
capable of directing transcription of the DNA sequence encoding the
polypeptide in the given host cell, (2) preferably, a 5'-untranslated
region (leader), (3) optionally, a signal sequence capable of directing
secretion of the polypeptide from the given host cell into the culture
medium, (4) the DNA sequence encoding a mature and preferably active form
of the polypeptide, and preferably also (5) a transcription termination
region (terminator) capable of terminating transcription downstream of
the DNA sequence encoding the polypeptide.

[0105]Downstream of the DNA sequence encoding the polypeptide, the
expression construct preferably contains a 3' untranslated region
containing one or more transcription termination sites, also referred to
as a terminator. The origin of the terminator is less critical. The
terminator can for example be native to the DNA sequence encoding the
polypeptide. However, preferably a yeast terminator is used in yeast host
cells and a filamentous fungal terminator is used in filamentous fungal
host cells. More preferably, the terminator is endogenous to the host
cell in which the DNA sequence encoding the polypeptide is expressed.

[0106]Enhanced expression of the polynucleotide encoding the polypeptide
of the invention may also be achieved by the selection of heterologous
regulatory regions, e.g., promoter, signal sequence and terminator
regions, which serve to increase expression and, if desired, secretion
levels of the protein of interest from the chosen expression host and/or
to provide for the inducible control of the expression of the polypeptide
of the invention.

[0107]Aside from the promoter native to the gene encoding the polypeptide
of the invention, other promoters may be used to direct expression of the
polypeptide of the invention. The promoter may be selected for its
efficiency in directing the expression of the polypeptide of the
invention in the desired expression host.

[0108]Promoters/enhancers and other expression regulation signals may be
selected to be compatible with the host cell for which the expression
vector is designed. For example prokaryotic promoters may be used, in
particular those suitable for use in E. coli strains. When expression of
the polypeptides of the invention is carried out in mammalian cells,
mammalian promoters may be used. Tissues-specific promoters, for example
hepatocyte cell-specific promoters, may also be used. Viral promoters may
also be used, for example the Moloney murine leukaemia virus long
terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter,
the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, herpes
simplex virus promoters or adenovirus promoters.

[0109]Suitable yeast promoters include the S. cerevisiae GAL4 and ADH
promoters and the S. pombe nmt1 and adh promoter. Mammalian promoters
include the metallothionein promoter which can be induced in response to
heavy metals such as cadmium. Viral promoters such as the SV40 large T
antigen promoter or adenovirus promoters may also be used. All these
promoters are readily available in the art.

[0116]The vector may further include sequences flanking the polynucleotide
giving rise to RNA which comprise sequences homologous to ones from
eukaryotic genomic sequences, preferably mammalian genomic sequences, or
viral genomic sequences. This will allow the introduction of the
polynucleotides of the invention into the genome of eukaryotic cells or
viruses by homologous recombination. In particular, a plasmid vector
comprising the expression cassette flanked by viral sequences can be used
to prepare a viral vector suitable for delivering the polynucleotides of
the invention to a mammalian cell. Other examples of suitable viral
vectors include herpes simplex viral vectors and retroviruses, including
lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses
(such as HPV-16 or HPV-18). Gene transfer techniques using these viruses
are known to those skilled in the art. Retrovirus vectors for example may
be used to stably integrate the polynucleotide giving rise to the
antisense RNA into the host genome. Replication-defective adenovirus
vectors by contrast remain episomal and therefore allow transient
expression.

[0117]The vector may contain a polynucleotide of the invention oriented in
an antisense direction to provide for the production of antisense RNA.
This may be used to reduce, if desirable, the levels of expression of the
polypeptide.

Host Cells and Expression

[0118]In a further aspect the invention provides a process for preparing a
polypeptide of the invention which comprises cultivating a host cell
transformed or transfected with an expression vector as described above
under conditions suitable for expression by the vector of a coding
sequence encoding the polypeptide, and recovering the expressed
polypeptide. Polynucleotides of the invention can be incorporated into a
recombinant replicable vector, such as an expression vector. The vector
may be used to replicate the nucleic acid in a compatible host cell. Thus
in a further embodiment, the invention provides a method of making a
polynucleotide of the invention by introducing a polynucleotide of the
invention into a replicable vector, introducing the vector into a
compatible host cell, and growing the host cell under conditions which
bring about the replication of the vector. The vector may be recovered
from the host cell. Suitable host cells include bacteria such as E. coli,
yeast, mammalian cell lines and other eukaryotic cell lines, for example
insect cells such as Sf9 cells and (e.g., filamentous) fungal cells.

[0119]Preferably the potypeptide is produced as a secreted protein in
which case the DNA sequence encoding a mature form of the polypeptide in
the expression construct is operably linked to a DNA sequence encoding a
signal sequence. In the case where the gene encoding the secreted protein
has in the wild type strain a signal sequence preferably the signal
sequence used will be native (homologous) to the DNA sequence encoding
the polypeptide. Alternatively the signal sequence is foreign
(heterologous) to the DNA sequence encoding the polypeptide, in which
case the signal sequence is preferably endogenous to the host cell in
which the DNA sequence is expressed. Examples of suitable signal
sequences for yeast host cells are the signal sequences derived from
yeast MFalpha genes. Similarly, a suitable signal sequence for
filamentous fungal host cells is, e.g., a signal sequence derived from a
filamentous fungal amyloglucosidase (AG) gene, e.g., the A. niger glaA
gene. This signal sequence may be used in combination with the
amyloglucosidase (also called (gluco)amylase) promoter itself, as well as
in combination with other promoters. Hybrid signal sequences may also be
used within the context of the present invention.

[0120]Preferred heterologous secretion leader sequences are those
originating from the fungal amyloglucosidase (AG) gene (glaA--both 18 and
24 amino acid versions, for example, from Aspergillus), the MFalpha gene
(yeasts, for example, Saccharomyces and Kluyveromyces) or the
alpha-amylase gene (Bacillus).

[0121]The vectors may be transformed or transfected into a suitable host
cell as described above to provide for expression of a polypeptide of the
invention. This process may comprise culturing a host cell transformed
with an expression vector as described above under conditions suitable
for expression of the polypeptide, and optionally recovering the
expressed polypeptide.

[0122]A further aspect of the invention thus provides host cells
transformed or transfected with or comprising a polynucleotide or vector
of the invention. Preferably the polynucleotide is carried in a vector
which allows the replication and expression of the polynucleotide. The
cells will be chosen to be compatible with the said vector and may for
example be prokaryotic (for example bacterial), or eukaryotic fungal,
yeast or plant cells.

[0123]The invention encompasses processes for the production of a
polypeptide of the invention by means of recombinant expression of a DNA
sequence encoding the polypeptide. For this purpose the DNA sequence of
the invention can be used for gene amplification and/or exchange of
expression signals, such as promoters, secretion signal sequences, in
order to allow economic production of the polypeptide in a suitable
homologous or heterologous host cell. A homologous host cell is herein
defined as a host cell which is of the same species or which is a variant
within the same species as the species from which the DNA sequence is
derived.

[0124]Suitable host cells are preferably prokaryotic microorganisms such
as bacteria, or more preferably eukaryotic organisms, for example fungi,
such as yeasts or filamentous fungi, or plant cells. In general, yeast
cells are preferred over filamentous fungal cells because they are easier
to manipulate. However, some proteins are either poorly secreted from
yeasts, or in some cases are not processed properly (e.g.,
hyperglycosylation in yeast). In these instances, a filamentous fungal
host organism should be selected.

[0125]Bacteria from the genus Bacillus are very suitable as heterologous
hosts because of their capability to secrete proteins into the culture
medium. Other bacteria suitable as hosts are those from the genera
Streptomyces and Pseudomonas. A preferred yeast host cell for the
expression of the DNA sequence encoding the polypeptide is one of the
genus Saccharomyces, Kluyveromyces, Hansenula, Pichia, Yarrowia, or
Schizosaccharomyces. More preferably, a yeast host cell is selected from
the group consisting of the species Saccharomyces cerevisiae,
Kluyveromyces lactis (also known as Kluyveromyces marxianus var. lactis),
Hansenula polymorpha, Pichia pastoris, Yarrowia lipolytica, and
Schizosaccharomyces pombe.

[0126]Most preferred for the expression of the DNA sequence encoding the
polypeptide are, however, filamentous fungal host cells. Preferred
filamentous fungal host cells are selected from the group consisting of
the genera Aspergillus, Trichoderma, Fusarium, Disporotrichum,
Penicillium, Acremonium, Neurospora, Thermoascus, Myceliophtora,
Sporotrichum, Thielavia, and Talaromyces. More preferably a filamentous
fungal host cell is of the species Aspergillus oyzae, Aspergillus sojae
or Aspergillus nidulans or is of a species from the Aspergillus niger
Group (as defined by Raper and Fennell, The Genus Aspergillus, The
Williams & Wilkins Company, Baltimore, pp. 293-344, 1965). These include
but are not limited to Aspergillus niger, Aspergillus awamori,
Aspergillus tubigensis, Aspergillus aculeatus, Aspergillus foetidus,
Aspergillus nidulans, Aspergillus japonicus, Aspergillus oryzae and
Aspergillus ficuum, and also those of the species Trichoderma reesei,
Fusarium graminearum, Penicillium chrysogenum, Acremonium alabamense,
Neurospora crassa, Myceliophtora thermophilum, Sporotrichum
cellulophilum, Disporotrichum dimorphosporum and Thielavia terrestris.

[0127]Examples of preferred expression hosts within the scope of the
present invention are fungi such as Aspergillus species (in particular
those described in EP 0184438 and EP. 0284603) and Trichoderma species;
bacteria such as Bacillus species (in particular those described in EP
0134048 and EP 0253455), especially Bacillus subtilis, Bacillus
licheniformis, Bacillus amyloliquefaciens, Pseudomonas species; and
yeasts such as Kluyveromyces species (in particular those described in
EP-A-096,430 such as Kluyveromyces lactis and in EP 0301670) and
Saccharomyces species, such as Saccharomyces cerevisiae.

[0128]Host cells according to the invention include plant cells, and the
invention therefore extends to transgenic organisms, such as plants and
parts thereof, which contain one or more cells of the invention. The
cells may heterologously express the polypeptide of the invention or may
heterologously contain one or more of the polynucleotides of the
invention. The transgenic (or genetically modified) plant may therefore
have inserted (typically stably) into its genome a sequence encoding the
polypeptides of the invention. The transformation of plant cells can be
performed using known techniques, for example using a Ti or a Ri plasmid
from Agrobacterium tumefaciens. The plasmid (or vector) may thus contain
sequences necessary to infect a plant, and derivatives of the Ti and/or
Ri plasmids may be employed.

[0129]The host cell may overexpress the polypeptide, and techniques for
engineering over-expression are well-known and can be used in the present
invention. The host may thus have two or more copies of the
polynucleotide.

[0130]Alternatively, direct infection of a part of a plant, such as a
leaf, root or stem can be effected. In this technique the plant to be
infected can be wounded, for example by cutting the plant with a razor,
puncturing the plant with a needle or rubbing the plant with an abrasive.
The wound is then innoculated with the Agrobacterium. The plant or plant
part can then be grown on a suitable culture medium and allowed to
develop into a mature plant. Regeneration of transformed cells into
genetically modified plants can be achieved by using known techniques,
for example by selecting transformed shoots using an antibiotic and by
sub-culturing the shoots on a medium containing the appropriate
nutrients, plant hormones and the like.

Culture of Host Cells and Recombinant Production

[0131]The invention also includes cells that have been modified to express
the proline-specific endoprotease or a variant thereof. Such cells
include transient, or preferably stably modified higher eukaryotic cell
lines, such as mammalian cells or insect cells, lower eukaryotic cells,
such as yeast and filamentous fungal cells or prokaryotic cells such as
bacterial cells.

[0132]It is also possible for the polypeptides of the invention to be
transiently expressed in a cell line or on a membrane, such as for
example in a baculovirus expression system. Such systems, which are
adapted to express the proteins according to the invention, are also
included within the scope of the present invention.

[0133]According to the present invention, the production of the
polypeptide of the invention can be effected by the culturing of
microbial expression hosts, which have been transformed with one or more
polynucleotides of the present invention, in a conventional nutrient
fermentation medium.

[0134]The recombinant host cells according to the invention may be
cultured using procedures known in the art. For each combination of a
promoter and a host cell, culture conditions are available which are
conducive to the expression the DNA sequence encoding the polypeptide.
After reaching the desired cell density or titre of the polypeptide the
culturing is ceased and the polypeptide is recovered using known
procedures.

[0136]The selection of the appropriate medium may be based on the choice
of expression host and/or based on the regulatory requirements of the
expression construct. Suitable media are well-known to those skilled in
the art. The medium may, if desired, contain additional components
favoring the transformed expression hosts over other potentially
contaminating microorganisms.

[0137]The fermentation may be performed over a period of from 0.5-30 days.
Fermentation may be a batch, continuous or fed-batch process, at a
suitable temperature in the range of between 0° C. and 45°
C. and, for example, at a pH from 2 to 10. Preferred fermentation
conditions include a temperature in the range of between 20° C.
and 37° C. and/or a pH between 3 and 9. The appropriate conditions
are usually selected based on the choice of the expression host and the
protein to be expressed.

[0138]After fermentation, if necessary, the cells can be removed from the
fermentation broth by means of centrifugation or filtration. After
fermentation has stopped or after removal of the cells, the polypeptide
of the invention may then be recovered and, if desired, purified and
isolated by conventional means. The proline-specific endoprotease of the
invention can be purified from fungal mycelium or from the culture broth
into which the proline-specific endoprotease is released by the cultured
fungal cells.

[0139]In a preferred embodiment the polypeptide is obtained from a fungus,
more preferably from an Aspergillus, most preferably from Aspergillus
niger.

Modifications

[0140]Polypeptides of the invention may be chemically modified, e.g.,
post-translationally modified. For example, they may be glycosylated (one
or more times) or comprise modified amino acid residues. They may also be
modified by the addition of histidine residues to assist their
purification or by the addition of a signal sequence to promote secretion
from the cell. The polypeptide may have amino- or carboxyl-terminal
extensions, such as an amino-terminal methionine residue, a small linker
peptide of up to about 20-25 residues, or a small extension that
facilitates purification, such as a poly-histidine tract, an antigenic
epitope or a binding domain.

[0141]A polypeptide of the invention may be labelled with a revealing
label. The revealing label may be any suitable label which allows the
polypeptide to be detected. Suitable labels include radioisotopes, e.g.,
125I , 35S, enzymes, antibodies, polynucleotides and linkers
such as biotin.

[0142]The polypeptides may be modified to include non-naturally occurring
amino acids or to increase the stability of the polypeptide. When the
proteins or peptides are produced by synthetic means, such amino acids
may be introduced during production. The proteins or peptides may also be
modified following either synthetic or recombinant production.

[0143]The polypeptides of the invention may also be produced using D-amino
acids. In such cases the amino acids will be linked in reverse sequence
in the C to N orientation. This is conventional in the art for producing
such proteins or peptides.

[0144]A number of side chain modifications are known in the art and may be
made to the side chains of the proteins or peptides of the present
invention. Such modifications include, for example, modifications of
amino acids by reductive alkylation by reaction with an aldehyde followed
by reduction with NaBH4, amidination with methylacetimidate or
acylation with acetic anhydride.

[0145]The sequences provided by the present invention may also be used as
starting materials for the construction of "second generation" enzymes.
"Second generation" proline-specific proteases are proline-specific
proteases, altered by mutagenesis techniques (e.g., site-directed
mutagenesis), which have properties that differ from those of wild-type
proline-specific protease or recombinant proline-specific proteases such
as those produced by the present invention. For example, their
temperature or pH optimum, specific activity, substrate affinity or
thermostability may be altered so as to be better suited for use in a
particular process.

[0146]Amino acids essential to the activity of the proline-specific
protease of the invention, and therefore preferably subject to
substitution, may be identified according to procedures known in the art,
such as site-directed mutagenesis or alanine-scanning mutagenesis. In the
latter technique mutations are introduced at every residue in the
molecule, and the resultant mutant molecules are tested for biological
activity (e.g., proline-specific endoprotease activity) to identify amino
acid residues that are critical to the activity of the molecule. Sites of
enzyme-substrate interaction can also be determined by analysis of
crystal structure as determined by such techniques as nuclear magnetic
resonance, crystallography or photo-affinity labelling.

[0147]The use of yeast and filamentous fungal host cells is expected to
provide for such post-translational modifications (e.g., proteolytic
processing, myristylation, glycosylation, truncation, and tyrosine,
serine or threonine phosphorylation) as may be needed to confer optimal
biological activity on recombinant expression products of the invention.

Preparations

[0148]Polypeptides of the invention may be in an isolated form. It will be
understood that the polypeptide may be mixed with carriers or diluents
which will not interfere with the intended purpose of the polypeptide and
still be regarded as isolated. A polypeptide of the invention may also be
in a substantially purified form, in which case it will generally
comprise the polypeptide in a preparation in which more than 70%, e.g.,
more than 80%, 90%, 95%, 98% or 99% of the proteins in the preparation is
a polypeptide of the invention.

[0149]Polypeptides of the invention may be provided in a form such that
they are outside their natural cellular environment. Thus, they may be
substantially isolated or purified, as discussed above, or in a cell in
which they do not occur in nature, for example a cell of other fungal
species, animals, plants or bacteria.

Removal or Reduction of Proline-Specific Endoprotease Activity

[0150]The present invention also relates to methods for producing a mutant
cell of a parent cell, which comprises disrupting or deleting the
endogenous nucleic acid sequence encoding the polypeptide or a control
sequence thereof, which results in the mutant cell producing less of the
polypeptide than the parent cell.

[0151]The construction of strains which have reduced proline-specific
endoprotease activity may be conveniently accomplished by modification or
inactivation of a nucleic acid sequence necessary for expression of the
proline-specific endoprotease in the cell. The nucleic acid sequence to
be modified or inactivated may be, for example, a nucleic acid sequence
encoding the polypeptide or a part thereof essential for exhibiting
proline-specific endoprotease activity, or the nucleic acid sequence may
have a regulatory function required for the expression of the polypeptide
from the coding sequence of the nucleic acid sequence. An example of such
a regulatory or control sequence may be a promoter sequence or a
functional part thereof, i.e., a part which is sufficient for affecting
expression of the polypeptide. Other control sequences for possible
modification include, but are not limited to, a leader sequence, a
polyadenylation sequence, a propeptide sequence, a signal sequence, and a
termination sequence.

[0152]Modification or inactivation of the nucleic acid sequence may be
performed by subjecting the cell to mutagenesis and selecting cells in
which the proline-specific endoprotease producing capability has been
reduced or eliminated. The mutagenesis, which may be specific or random,
may be performed, for example, by use of a suitable physical or chemical
mutagenizing agent, by use of a suitable oligonucleotide, or by
subjecting the DNA sequence to PCR mutagenesis. Furthermore, the
mutagenesis may be performed by use of any combination of these
mutagenizing agents.

[0154]When such agents are used, the mutagenesis is typically performed by
incubating the cell to be mutagenized in the presence of the mutagenizing
agent of choice under suitable conditions, and selecting for cells
exhibiting reduced or no expression of proline-specific endoprotease
activity.

[0155]Modification or inactivation of production of a polypeptide of the
present invention may be accomplished by introduction, substitution, or
removal of one or more nucleotides in the nucleic acid sequence encoding
the polypeptide or a regulatory element required for the transcription or
translation thereof. For example, nucleotides may be inserted or removed
so as to result in the introduction of a stop codon, the removal of the
start codon, or a change of the open reading frame. Such modification or
inactivation may be accomplished by site-directed mutagenesis or PCR
mutagenesis in accordance with methods known in the art.

[0156]Although, in principle, the modification may be performed in
vivo--i.e., directly on the cell expressing the nucleic acid sequence to
be modified--it is preferred that the modification be performed in vitro
as exemplified below.

[0157]An example of a convenient way to inactivate or reduce production of
the proline-specific endoprotease by a host cell of choice is based on
techniques of gene replacement or gene interruption. For example, in the
gene interruption method, a nucleic acid sequence corresponding to the
endogenous gene or gene fragment of interest is mutagenized in vitro to
produce a defective nucleic acid sequence which is then transformed into
the host cell to produce a defective gene. By homologous recombination,
the defective nucleic acid sequence replaces the endogenous gene or gene
fragment. Preferably the defective gene or gene fragment also encodes a
marker which may be used to select for transformants in which the gene
encoding the polypeptide has been modified or destroyed.

[0158]Alternatively, modification or inactivation of the nucleic acid
sequence encoding a polypeptide of the present invention may be achieved
by established anti-sense techniques using a nucleotide sequence
complementary to the polypeptide encoding sequence. More specifically,
production of the polypeptide by a cell may be reduced or eliminated by
introducing a nucleotide sequence complementary to the nucleic acid
sequence encoding the polypeptide. The antisense polynucleotide will then
typically be transcribed in the cell and will be capable of hybridizing
to the mRNA encoding the proline-specific endoprotease. Under conditions
allowing the complementary antisense nucleotide sequence to hybridize to
the mRNA, the amount of the proline-specific endoprotease produced in the
cell will be reduced or eliminated.

[0159]It is preferred that the cell to be modified in accordance with the
methods of the present invention is of microbial origin, for example, a
fungal strain which is suitable for the production of desired protein
products, either homologous or heterologous to the cell.

[0160]The present invention further relates to a mutant cell of a parent
cell which comprises a disruption or deletion of the endogenous nucleic
acid sequence encoding the polypeptide or a control sequence thereof,
which results in the mutant cell producing less of the polypeptide than
the parent cell.

[0161]The polypeptide-deficient mutant cells so created are particularly
useful as host cells for the expression of homologous and/or heterologous
polypeptides. Therefore, the present invention further relates to methods
for producing a homologous or heterologous polypeptide comprising (a)
culturing the mutant cell under conditions conducive for production of
the polypeptide; and (b) recovering the polypeptide. In the present
context, the term "heterologous polypeptides" is defined herein as
polypeptides which are not native to the host cell, a native protein in
which modifications have been made to alter the native sequence, or a
native protein whose expression is quantitatively altered as a result of
a manipulation of the host cell by recombinant DNA techniques.

[0162]In a still further aspect, the present invention provides a method
for producing a protein product essentially free of proline-specific
endoprotease activity by fermentation of a cell which produces both a
proline-specific endoprotease polypeptide of the present invention as
well as the protein product of interest. The method comprises adding an
effective amount of an agent capable of inhibiting proline-specific
endoprotease activity to the fermentation broth either during or after
the fermentation has been completed, recovering the product of interest
from the fermentation broth, and optionally subjecting the recovered
product to further purification. Alternatively, after cultivation the
resultant culture broth can be subjected to a pH or temperature treatment
so as to reduce the proline-specific endoprotease activity substantially,
and allow recovery of the product from the culture broth. The combined pH
or temperature treatment may be performed on a protein preparation
recovered from the culture broth.

[0163]The methods of the present invention for producing an essentially
proline-specific, endoprotease-free product is of particular interest in
the production of eukaryotic polypeptides, in particular in the
production of fungal proteins such as enzymes. The proline-specific
endoprotease-deficient cells may also be used to express heterologous
proteins of interest for the food industry, or of pharmaceutical
interest.

[0164]Preferred sources for the proline-specific endoprotease are obtained
by cloning a microbial gene encoding a proline-specific endoprotease into
a microbial host organism. More preferred sources for the
proline-specific endoprotease are obtained by cloning an
Aspergillus-derived gene encoding a proline-specific endoprotease into a
host belonging to the genus of Aspergillus capable of overexpressing the
proline-specific endoprotease gene.

[0165]In the category of products containing protein hydrolysates
targeting consumers with non-medical needs, the niche market of employing
protein hydrolysates in products for athletes is rapidly increasing. In
this product category, the allergenicity of the final product is not an
issue. Instead, aspects such as taste, nutritional value and the presence
of specific amino acids to support endurance and stimulate physiological
recovery after exercise are important parameters for such hydrolysates,
particularly when used in sport drinks. For example, glutamine has been
implicated in fighting metabolic stresses but can only be supplied in
small peptides, as the free amino acid is not stable in solution. Protein
hydrolysates produced according to the invention are very suitable for
use in athletic-related products due to their very high solubility under
the acid pH conditions prevalent, for example, in sport drinks. An
important implication of this criterion is that high levels of
hydrolysates produced according to the invention can be included in
nutritional sport products without the drawback of protein precipitation
upon sterilisation and prolonged storage. Thus, the shelf lives of sport
products may be extended by the addition of a protein hydrolysate of the
invention. The enzyme mixture according to the invention may be used to
hydrolyze proteinaceous materials of animal origin such as whole milk,
skim milk, casein, whey protein or mixtures of casein and whey protein.
Such mixtures of casein and whey protein may be used, for example, in
ratios similar to those found in human milk. Furthermore, collagen based
animal proteins forms a substrate because of the possibility to degrade
these proteins to smaller molecules hereby debittering animal meat
extracts or improving the uptake of proline and hydroxyproline residues
with benefits on the joints of athletes.

[0166]The enzyme mixture according to the invention may also be used to
hydrolyze proteinaceous materials of plant origin such as, for example,
wheat gluten malted or unmalted barley or other cereals used for making
beer, soy milk, concentrates or isolates thereof, maize protein
concentrates and isolates thereof, and rice proteins.

[0167]The invention will be further illustrated by the following
non-limiting Examples.

Examples

Materials & Methods

[0168]Beta-casein from bovine milk (lyophilized, essentially salt-free
powder) with a minimum of 90% beta-casein was obtained from Sigma.
Collagen (type 1, insoluble from bovine achilles tendon) was also
obtained from Sigma.

[0170]A low bitterness whey hydrolysate VITALARMOR® 800 LB as well as
whey protein enriched in beta-lactoglobulin (PROTARMOR® 905) was
obtained from Armor Proteines (Saint-Brice-en-Cogles, France). Other
commercial hydrolysates were obtained from the producer or purchased in
pharmacies.

[0173]Proline-specific endoprotease from Flavobacterium meningosepticum
and cloned in E. coli was isolated using known plasmid constructs and
enzyme purification methods (T. Diefenthal and H. Dargatz, World Journal
of Microbiology & Biotechnology 11: 209-212, 1995) The enzymatic activity
was tested on CBZ-Gly-Pro-pNA 0.26 mM in phosphate buffer 0.1M pH 7.0 at
25° C. pH 7.0 was used in this test because the pH optimum of this
enzyme is above pH 6.0. The product was monitored spectrophotometrically
at 410 nm.

[0174]A unit was defined as the quantity of enzyme that provokes the
release of 1 μmol of p-nitroanilide per minute under these conditions.

[0175]Proline-specific endoproteases from Aspergilli were measured
according to the method described in JP 05-015314 with minor
modifications. In brief the enzymatic activity is tested on
CBZ-Gly-Pro-pNA at 37° C. in a citrate/disodium phosphate buffer
pH 5. pH 5.0 is chosen because in this test the pH optimum of the enzyme
is below pH 6. The reaction product was also monitored spectrophoto
metrically at 410 nM.

[0177]Proline-specific endoprotease from A. niger G-306 was produced and
isolated as outlined in Example 4. Complete purification was realised
using two-dimensional gel electrophoresis. To that end the active
material isolated from the SUPERDEX® 75 column was first desalted by
dilution (approx 20 fold) in 10 mM Tris/HCl buffer pH 6.8 and then
concentrated with a CENTRICON® 30 kD miniconcentrator (Amicon).

[0178]Basically the two-dimensional electrophoresis was performed as
described in 2-D Electrophoresis Using Immobilized pH Gradients, Amersham
Pharmacia Biotech 80-6429-60 Rev A/10-98. The first dimension (IEF) was
performed on an IPGphor (Amersham-Pharmacia) using a 11 cm IPG strip pH
range 3-6 (BioRad) The desalted, 3-fold concentrated sample was diluted
in 8M urea (6M urea and 2M thiourea). This was mixed with 18.5
microliters of 10×-concentrated rehydration buffer, containing 6M
urea, 2M thiourea 20% CHAPS, and 5% IPG buffer range 3-10. The total was
used to rehydrate the IPG strip. Focussing was done during 29.320 Vh
using the protocol as described in the Biorad leaflet supplied with the
strips as a guideline.

[0179]The second dimension (SDS) was done on a Criterion Mini Vertical
Cell (BioRad) using a precast gel of 12% (Type Prep+2 Comb) purchased
from BioRad. The IPG strip was first incubated in SDS equilibration
buffer containing DTT (1%) and a second time in buffer containing
iodoacetamide (2.5%) Both incubations were for 15 min at 20° C.
The SDS equilibration buffer consisted of Tris/HCl 50 mM pH 8.8, 6M urea,
30% (v/v) glycerol and 2% (w/v) SDS and a trace of bromophenol blue.

[0180]After incubation the IPG strip was trimmed to fit the gel type
mentioned and ran with 10× diluted TGS buffer (BioRad). After the
run the gel was stained with Sypro Ruby (Molecular Probes, Leiden,
Netherlands) for 3-4 hr and washed with Milli Q water for 2 hr. Imaging
was performed on The Imager (Appligene). The largest spot was cut out,
washed several times with 50 mmol/liter ammonium bicarbonate, incubated
overnight at 37° C. with sequencing grade trypsin (nr. 1047841,
Boehringer Mannheim). Peptides were extracted from the gel piece by
washing several times with acetonitrile/water containing formic acid
(50/50/5, v/v/v). The samples were dried using a vacuum centrifuge (New
Brunswick Scientific, Netherlands) and stored at -20° C., until
analysis.

LC/MS Analysis

[0181]HPLC (high performance liquid chromatography) using a Qtof-2
(Micromass, Manchester, UK) mass spectrometer was used to separate the
peptides formed during digestion with trypsin. Five microliters of the
peptide solution was trapped on a micro-precolumn, C18, 5*0.3 mm
(MCA30-05-C18, LC Packings, Amsterdam, Netherlands) using Milli Q water
containing 0.1% of formic acid at a flow-rate of 20 microliter/min. The
peptides were then eluted from the precolumn, using a fast gradient of
0.1% formic acid in Milli Q water (Millipore, Bedford, Mass., USA;
Solution A) and 0.1% formic acid in acetonitrile (Solution B). The
gradient started at 100% of Solution A and increased to 60% of solution B
in 20 min and was kept at the latter ratio for another 5 min. The flow
rate used during elution of the peptides was 200 nl/min. Using LC/MS/MS
analysis partial amino acid sequences of the A. niger proline-specific
endopeptidase could be determined, by de novo sequencing of suitable
peptides.

[0182]HPLC using an ion trap mass spectrometer (THERMOQUEST®, Breda,
Netherlands) coupled to a P4000, pump (THERMOQUEST®, Breda,
Netherlands) was used in characterizing the enzymatic protein
hydrolysates produced by the inventive enzyme mixture. The peptides
formed were separated using a PEPMAP C18 300A (MIC-15-03-C18-PM, LC
Packings, Amsterdam, Netherlands) column in combination with a gradient
of 0.1% formic acid in Milli Q water (Millipore, Bedford, Mass., USA;
Solution A) and 0.1% formic acid in acetonitrile (Solution B) for
elution. The gradient started at 100% of Solution A and increased to 70%
of solution B in 45 min and was kept at the latter ratio for another 5
min. The injection volume used was 50 microliters, the flow rate was 50
microliters per minute and the column temperature was maintained at
30° C. The protein concentration of the injected sample was
approx. 50 microgram/ml.

[0183]Detailed information on the individual peptides was obtained by
using the "scan dependent" MS/MS algorithm which is a characteristic
algorithm for an ion trap mass spectrometer.

[0184]Full scan analysis was followed by zoom scan analysis for the
determination of the charge state of the most intense ion in the full
scan mass range. Subsequent MS/MS analysis of the latter ion resulted in
partial peptide sequence information, which could be used for database
searching using the SEQUEST application from Xcalibur Bioworks
(THERMOQUEST®, Breda, Netherlands). Databanks used were extracted from
the OWL.fasta databank, available at the NCB! (National Centre for
Biotechnology informatics), containing the proteins of interest for the
application used. In those experiments in which well characterized
protein substrates such as whey proteins or caseins were measured, the
precision of the analysis technique was increased by omitting those MS/MS
spectra with a sequence fit of less than 50%.

[0185]Only peptides with a mass ranging from approx. 400 to 2000 Daltons
were considered suitable for further analysis by MS sequencing.

[0186]Angiotensin (M=1295.6) was used to tune for optimal sensitivity in
MS mode and for optimal fragmentation in MS/MS mode, performing constant
infusion of 60 μg/ml, resulting in mainly doubly and triply charged
species in MS mode, and an optimal collision energy of about 35% in MS/MS
mode.

LC/MS Analysis of Infant Formulae and Commercial Protein Hydrolysates

[0187]Prior to LC/MS fatty material had to be removed from the infant
formulae. To that end the complete nutrition samples (13.5 g powder in
100 ml MilliQ water) were extracted 3 times with 30 ml hexane. Small
amounts of NaCl were added to improve separation of the solvent layers.
Then 5 ml of the water layer was obtained and freeze dried. Prior to
analysis the sample was redissolved in 25 ml of MilliQ water,
centrifugated 2 times (at 13000 rpm) and filtered through a 0.22 μm
filter. From pure hydrolysated samples, 400 mg was dissolved in 100 ml
MilliQ water, centrifugated 2 times (at 13000 rpm) and filtered through a
0.22 μm filter. To characterize the peptides present in the commercial
protein hydrolysates, the same strategy was followed as described above
for the enzymatic hydrolysates formed by the inventive enzyme mixture,
i.e., the filtered hydrolysate was applied to the HPLC column and
individual peptides with molecular masses between 400 and 2000 daltons
were further characterized by the MS/MS analysis. However, the databank
used to obtain peptide sequence information on whey or casein derived
hydrolysates consisted of cow milk protein sequences only.

[0188]LC/MS/MS can be used for the analysis of the C-terminus of a
peptide. With an algorithm in which the peptide's molecular mass
(analyzed with LC/MS) and its (partial) amino acid sequence (analyzed
with LC/MS/MS) are linked with automatic search procedures within protein
databanks, complex peptide mixtures can be analyzed. These options have
enabled us to quantify the incidence of peptides carrying a
carboxy-terminal proline residue. Owing to the limitations set by the
PEPMAP peptide separation column used, only peptides with a molecular
weight between roughly 400 and 2000 Dalton are analyzed using this
technique. Fortunately, in protein hydrolysates the majority of the
peptides have such molecular weights.

[0189]To determine in a protein hydrolysate the molar fraction of peptides
carrying a carboxy-terminal proline, individual peptide peaks eluting
from the PEPMAP column are selected and partial carboxy-terminal amino
acid sequences are determined using the techniques specified above.
Analysis of at least 20, preferably at least 30 and more preferably
between 40 to 60, for example 50 of the most abundant, randomly choosen
peptides thus provides insight in the frequency in which peptides
carrying a proline residue at the carboxy-terminus of the peptide occur.
The quotient of the number of peptides found to carry a carboxy-terminal
proline residue times 100 and the total number of peptides analyzed thus
provides the molar fraction of peptides (%) carrying a carboxy-terminal
proline.

Determination of Molar Fraction (%) of Proline in the Protein Substrate
Used to Generate the Hydrolysate

[0190]Fatty material as can occur in infant formulae products was first
removed by hexane extraction as detailed in the paragraph describing
LC/MS analysis of infant formulae and commercial protein hydrolysates.
Acid hydrolysis of the protein substrate to convert the proteins present
into free amino acids, was achieved by making a suspension of 100 mg of
proteinaceous material in 2 ml of 6N HCl. Acid hydrolysis was carried out
for 22 hr at 112° C. in an oxygen free atmosphere. After
centrifugation the supernatant was diluted 10 times in dilute HCl. After
this hydrolysis the amino acids were derivatized and analyzed according
to the Picotag method as specified in the operator's manual of the Amino
Acid Analysis System of Waters (Milford Mass., USA). The level of proline
present was quantitated using HPLC methods. To determine the molar
fraction (%) of proline in the sample, the micromoles of proline present
times 100 were divided by the sum of the micromoles of all amino acids
present in the sample analyzed. Since during acid hydrolysis Trp and Cys
are destroyed, these two amino acids are not included in this sum of the
micromoles of all amino acids.

[0191]A precisely weighed sample of the proteinaceous material was
dissolved in dilute acid and precipitates were removed by centrifugation
in an Eppendorf centrifuge. Amino acid analysis was carried out on the
clear supernatant according to the PicoTag method as specified in the
operators manual of the Amino Acid Analysis System of Waters (Milford
Mass., USA). To that end a suitable sample was obtained from the liquid,
added to dilute acid and homogenized. From the latter solution a new
sample was taken, dried and derivatized using phenylisothiocyanate. The
various derivatized amino acids present were quantitated using HPLC
methods and added up to calculate the total level of free amino acids in
the weighed sample.

[0192]To relate this total level of free amino acids in the sample to the
total level of amino acids that can be liberated from this sample, the
sample is also subjected to acid hydrolysis followed by a quantification
of the total free amino acids present as detailed above.

Example 1

[0193]Hydrolysis of Beta-Casein Using Subtilisin in Combination with a
Proline-Specific Endoprotease from F. meningosepticum

[0194]Beta-casein represents one of the major casein fractions of bovine
milk. The protein has been well characterized in terms of its amino acid
sequence and is commercially available in an almost pure form. As such,
beta-casein offers an excellent test substrate for studying the
relationship between enzyme cleavage sites and the length of various
peptides formed during enzyme hydrolysis.

[0195]This Example demonstrates that despite the broad spectrum character
of subtilisin, the addition of a very specific enzyme like a
proline-specific endoprotease can have a major impact on the size of the
beta-casein fragments formed. Improved yields for casein fractions upon
incubation with subtilisin in combination with a proline-specific
endoprotease can therefore be obtained. Beta-casein is relatively rich in
proline as acid hydrolysis followed by amino acid analysis carried out
according to the Materials & Methods section revealed that its molar
fraction of proline is 14% (moles of proline/moles of all amino acids as
specified in the Materials&Methods section).

[0196]Beta-casein powder (Sigma) was dissolved at a concentration of 10%
(w/w) together with 0.1% (w/w) DELVOLASE® in a 0.1 mol/liter
phosphate buffer pH 7.0. After an incubation of 24 hr at 45° C. in
a shaking waterbath, the reaction was stopped by heating the solution for
15 min at 90° C. To one-half of the solution (1 ml containing 100
mg of beta-casein) 100 microliter of proline-specific endoprotease from
F. meningosepticum (corresponding to 4 units according to the procedure
described in World Journal of Microbiology & Biotechnology, 11, 209-212
(1995) was added and the reaction was continued for another 24 hr at
45° C. After another heat shock at 90° C., samples of both
the DELVOLASE® and the DELVOLASE® +proline-specific endoprotease
treated beta-casein material were analyzed by LC/MS equipment as
specified in the Materials & Methods section.

[0197]In the sample digested with DELVOLASE® alone, the LC/MS/MS
analysis identified 40 peptides covering various parts of the beta-casein
molecule. Together these peptides accounted for 79% of the total
beta-casein sequence. Different retention times of the peptides on the
C18 column could be traced back to peptide lengths ranging from 2 to 23
amino acid residues. Glutamine proved to be the most frequently occurring
carboxy-terminal residue (10 out of 40 peptides). None of the peptides
analyzed could be shown to have proline as the carboxy-terminal residue.

[0198]By contrast, the sample digested with DELVOLASE® and
proline-specific endoprotease generated 28 identifiable peptides from
beta-casein. Together these peptides covered 63% of the total beta-casein
protein sequence. Peptide size distribution was remarkably homogeneous,
as the peptides ranged in length only between 3 and 9 residues. Within
this peptide population, glutamine was the carboxy-terminal residue in 3
peptides only and proline proved to be the most abundant carboxy-terminal
residue (in 17 out of the 28 peptides analyzed). The results show that in
the hydrolysate made with the proline-specific endoprotease, those
peptides that carry a carboxy-terminal proline residue represent a molar
fraction of 61% of the total of the peptides present in the molecular
weight range between 400 and 2000 daltons. Thus, incubation of
beta-casein with a proline-specific-endopeptidase results in the
generation of peptides with proline as the carboxy-terminal residue.
Moreover, the combination of subtilisin plus a proline-specific
endoprotease results in a remarkably homogeneous size distribution of the
various peptides generated, suggesting high product yields upon
ultrafiltration of such a hydrolysate.

Example 2

Beta-Casein Hydrolysates and Bitterness

[0199]Although Example 1 illustrates the effect of a proline-specific
endoprotease on peptide size and the proportion of peptides with proline
as the carboxy-terminal amino acid residue, the effect of this enzyme on
bitterness was not measured in Example 1. Casein hydrolysates are
notoriously bitter and this property has been linked to their relatively
high content of hydrophobic amino acid residues.

[0200]To test the effect of a proline-specific endoprotease on the taste
of beta-casein hydrolyzed by a subtilisin, enzyme incubations using
DELVOLASE® and the DELVOLASE® with proline-specific endoprotease
were performed as described in Example 1. Following heat inactivation of
both subtilisin and proline-specific endoprotease, samples were cooled to
room temperature and distilled water was added to give final casein
concentrations of 4% (w/w). The taste of the latter solutions were then
evaluated by a panel of experienced tasters. The tasters were unanimous
in their conclusion that the hydrolysate obtained by the combination of
subtilisin plus proline-specific endoprotease was significantly less
bitter than the hydrolysate obtained using subtilisin alone.

[0201]Thus, the treatment of casein hydrolysates with a proline-specific
endoprotease substancially reduces the bitterness of the final product.

[0203]A large collection of moulds capable of forming black spores were
allowed to grow in a pH 6.5 medium containing 1.0 gram of K2HPO4, 0.5
gram of KH2PO4, 0.5 gram of KCl, 0.5 gram of MgSO4. 7H2O, 0.01 gram of
FeSO4. 7H2O, 5 grams of glucose, 15 grams of collagen (Sigma) and
distilled water added to obtain a volume of 1 liter. The inoculum for
each experiment was prepared by a method in which the spores of fungi
growing on an agar slant (5 days old) were taken up in 5 ml of sterile
water. Of the latter suspension, 2% (v/v) was used for inoculation of the
pH 6.5 medium. Growth was allowed for 100 hr at 28° C. with
shaking after which the culture was filtrated and samples of the clear
filtrate were incubated with the synthetic peptide Z-Ala-Pro-pNA (Bachem;
Bubendorf, Switzerland) at pH 5.0 and 50° C. Samples capable of
releasing pNA were identified by measuring the increase in absorbance at
410 nanometer. Positive strains yielding relatively high activities were
further investigated.

[0204]Strain G-306 excreted a proline-specific endoprotease and was
identified as Aspergillus niger Van Tieghem var. niger. This particular
strain was used for isolation, purification and further characterization
of a proline-specific endoprotease. To purify the enzyme 1 liter of
culture supernatant was applied to a 400 ml bacitracin-silochrome column
equilibrated with 0.05 mol/liter sodium acetate pH 5.0. Proteases bound
to the column were eluted using the acetate buffer supplemented with 1
mol/liter of NaCl and 10% (v/v) isopropanol (J. Appl. Biochem., pp.
420-428, 1983). Active fractions were collected and dialysed against
distilled water and applied on a 200 ml bacitracin-Sepharose column,
again equilibrated with acetate buffer. As before, elution was carried
out using the acetate buffer supplemented with NaCl and isopropanol.
Active fractions were collected, dialysed against a 5 mmol/liter acetate
buffer pH 5.0 and then concentrated by means of ultrafiltration with a
Amicon PM-10 membrane. To obtain an almost completely pure
proline-specific endoprotease, the concentrated liquid was
chromatographed over a SUPERDEX® 75 column eqiulibrated with the 0.05
mol/liter sodium acetate buffer pH 5.0 and supplemented with 0.5
mol/liter NaCl.

[0205]Further experiments carried out with the purified enzyme indicated a
molecular weight around 66.6 kDalton, an IEP around pH 4.2, a pH optimum
around 5.0 and an almost 100% thermostablity upon incubation for 4 hr at
50° C.

[0206]To obtain partial amino acid sequences of the enzyme, the enzyme
preparation isolated was first subjected to two-dimensional gel
electrophoresis according to the procedure described in the Materials &
Methods section. The largest spot was cut out, incubated with trypsin and
eluted. The recovered peptides were then subjected to LC/MS/MS analysis
as described in the Materials & Methods section to determine partial
amino acid sequences.

[0207]The following amino acid sequences could be derived from the
proline-specific endoprotease of Aspergillus niger

These amino acid sequences were used to synthesize the DNA sequences
needed for the isolation of the gene encoding the proline-specific
endoprotease from Aspergillus niger.

[0208]In later experiments (see Example 10) the sequence
NH2-ATTGEAYFE-COOH could be shown to represent the amino-terminus of the
mature proline-specific endoprotease.

Example 4

Proline-Specific Endoprotease and its Effects in Hydrolysis of Soy Protein

[0209]Japanese patent JP 05-01314 describes a crude enzyme preparation
obtained from Aspergillus oryza FS1-32 that exhibits major quantities of
a non-specific endoproteolytic activity and minor quantities of a
proline-specific endoprotease and a carboxypeptidase activity. Incubation
of soy bean protein with this crude enzyme preparation is claimed to
yield a hydrolysate that is significantly less bitter than a soy bean
hydrolysate that can be obtained with another protease preparation which
lacks a proline-specific endoprotease in combination with a
carboxypeptidase. It is suggested in JP 05-015314 that the activity of
the proline-specific endoprotease exposes proline residues that are
subsequently removed by the carboxypeptidase. The removal of these
hydrophobic, carboxy-terminal proline residues by the carboxypeptidase is
thought to be essential for obtaining less bitter hydrolysates.

[0210]To test this statement, one of the Examples provided in JP 05-015314
was repeated and the resulting soy hydrolysates were analyzed using the
above described LC/MS technology rather than evaluating an effect on
taste.

[0211]According to JP 05-015314, their incubations with Aspergillus oryzae
FS 1-32 contained per gram of substrate the following enzymatic
activities.

[0212]Protease: in the order of 650 PU; carboxypeptidase: in the order of
0.01 unit and proline-specific endopeptidase: in the order of 0.03
milli-Units.

[0213]Because the original Aspergillus oryzae FS 1-32 preparation was not
available, two commercial enzyme preparations, also derived from
Aspergillus oryzae, were used in the present Example. Moreover, a
chromatographically purified proline-specific endoprotease isolated from
Aspergillus niger (see Example 3) was used to achieve an overdosing of
the acid proline-specific endoprotease.

[0214]The enzymatic activities of the various preparations were measured
according to the procedures provided in JP 05-015314 and are provided
below. [0215]SUMIZYME® LP 75.000, a commercial Aspergillus oryzae
enzyme preparation known to be rich in endoproteolytic activity.
[0216]Enzymatic activities as assessed according to the methods of JP
05-015314: [0217]Protease: 226 PU/gram product; carboxypeptidase: 21
units/gram product; prolyl-endopeptidase: 430 milli-Units/gram product
[0218]FLAVOURZYME® 1000L, a commercial Aspergillus oryzae enzyme
preparation known to be rich in exoproteolytic activity. [0219]Enzymatic
activities as assessed according to the methods of JP 05-015314:
[0220]Protease: 332 PU/gram product; carboxypeptidase: 10 units/gram
product; prolyl-endopeptidase: not detectable [0221]Chromatographically
pure proline-specific endoprotease obtained from Aspergillus niger and
isolated as described in Example 3.

[0222]Enzymatic activities as assessed according to the methods of JP
05-015314: Protease: not detectable; carboxypeptidase: not detectable;
prolyl-endopeptidase: 45 milli-Units/ml.

[0223]From the data it is evident that although SUMIZYME® and
FLAVOURZYME® are well-known for their high proteolytic activities,
none of them can provide the same very high ratio of (endo)protease to
carboxypeptidase activity as quoted in JP 05-015314. Surprisingly
SUMIZYME® LP 75.000 was found to contain a considerably higher
activity of proline-specific endoprotease than the one reported in JP
05-015314.

[0224]The various enzyme preparations were incubated according to the
protocol described in JP 05-015314 but standardized according to the
desired carboxypeptidase activity (0.01 unit per gram substrate). Soy
isolate (SOYAMIN® 90 HV) was used as the substrate in these
reactions. After incubation for 5 hr at pH 5 and 50° C., the
samples were centrifuged and the supernatants were kept frozen until
LC/MS analysis.

[0225]LC/MS analysis was carried out as specified in the Materials &
Methods section.

[0226]In this experiment the protein data bank consisted of soy proteins
only. The results obtained are specified in Table 1.

[0227]SUMIZYME® LP 75.000 contains a proline-specific endoproteolytic
activity which is about 7 times higher than the proline-specific
endoproteolytic activity recorded in strain FS 1-32 and yields a molar
fraction of approx 10% of soy peptides carrying a carboxy-terminal
proline. SUMIZYME® LP 75.000 enriched with the proline-specific
endoprotease isolated from Aspergillus niger contains a proline-specific
endoproteolytic activity which is about 50 times higher than the activity
recorded with strain FS 1-32 but also yields a molar fraction of approx
10% of soy peptides carrying a carboxy-terminal proline. These data were
confirmed by analysing the number of proline residues which are present
in the peptides but not in the carboxy-terminal position.
FLAVOURZYME® contains no detectable proline-specific endpoprotease
but yields among the peptides generated and suitable for analysis with
the LC/MS technique a molar fraction of 6% of peptides carrying a proline
at the carboxy-terminal end. If combined with a proline content of
approx. 5% of this soy protein isolate, these three observations indicate
that the presence and the activity of the proline-specific endoprotease
in combination with the carboxypeptidase activity has a minor effect on
the molar incidence of carboxy-terminal proline residues only. So, it is
hard to imagine that the debittering effect described in JP 05-015314 and
ascribed to a proline-specific endoprotease activity of 0.03 milli-Units
only can be linked to a high incidence of peptides carrying proline as
the carboxy-terminal amino acid residue.

Example 5

Increased Dosages of Proline-Specific Endoprotease and its Effects on
Hydrolysis of Soy Protein

[0228]In this Example it is demonstrated that high levels of a
proline-specific endoprotease are required to generate soy hydrolysates
containing a significant amount of peptides carrying a carboxy-terminal
proline residue. The overall design of these experiments was identical to
the ones described in Example 4. Again soy protein isolate was incubated
with SUMIZYME® LP 75.000 standardized according to the desired
carboxypeptidase activity of 0.01 unit per gram soy protein and under
conditions described in JP 05-015314. The incubation took place for
either 2.5 or 5.0 hr at pH 5 and 50° C. and was stopped by keeping
the material for 10 min at 100° C. Subsequently some of the
material incubated for 5 hr was obtained and its pH was increased to 7.0.
From this material, three samples were obtained to which different
portions of the E. coli produced F. meningosepticum proline-specific
endoprotease were added. To the first sample 1.5 milli-Units of
proline-specific endoprotease (according to JP 05-015314 but measured at
pH 7.0 and 30° C. to accommodate the pH and temperature optimum of
the E. coil derived proline-specific endoprotease) were added, to the
second sample 150 milli-Units were added and to the third sample 15,000
milli-Units were added and then the samples were again incubated for 2 hr
at 40° C. After incubation the samples were centrifuged and the
supernatants were kept frozen until LC/MS analysis. LC/MS analysis took
place as specified earlier. The results obtained are specified in Table
2.

[0229]The results obtained clearly illustrate that a significant increase
in the incidence of peptides carrying a carboxy-terminal proline residue
in the hydrolysate is totally dependent upon the addition of the
proline-specific endoprotease. However, only activities which exceed the
activity mentioned in JP 05-015314 and the activity present in
SUMIZYME® LP 75 000 by several orders of magnitude are capable of
doing this. The implication of this observation is that a pure and
isolated proline-specific endoprotease is essential to obtain the desired
peptide composition of the hydrolysate.

[0230]As described earlier, LC/MS/MS can be used for the analysis of the
C-terminus of a peptide. With an algorithm in which the peptide's
molecular mass (analyzed with LC/MS) and its (partial) amino acid
sequence (analyzed with LC/MS/MS) are linked with automatic search
procedures within protein databanks, complex peptide mixtures can be
analyzed.

[0231]In this Example these possibilities were used to analyze a number of
commercial infant formulae products as well as commercial protein
hydrolysates for the molar incidence of peptides carrying
carboxy-terminal proline residues which have a molecular weight between
400 and 2000 daltons.

[0243]As the infant formulae contain approx. 15% of protein hydrolysate
plus fats (25%) and carbohydrates (50%), a hexane extraction of these
products to remove the fat phase proved to be indispensible. The pure
hydrolysates could be used as such.

[0244]To link the partial protein sequences obtained with sequences of
known proteins, a databank containing cow milk protein sequences only was
used for all samples except the PREGOMIN® sample. The PREGOMIN®
sample was analyzed using a databank containing soy- and
collagen-specific sequence data. For analytical reasons the LC/MS
analysis focusses on peptides with a molecular weight ranging from 400 to
approx. 2000 Daltons so that peptides outside this range are not taken
into consideration.

[0245]In each sample between 32 and 76 peptides containing sequence
information of the hydrolyzed proteins used could be identified. In most
samples more than 95% of the 25 most intense peaks in the chromatogram
could be related to sequence information of milk proteins. In the
PREGOMIN® sample only 65% of the 25 most intense peaks could be
related to sequence information of soy and collagen proteins. Possible
reasons for this are the incorporation of other protein sources in the
protein basis or poor MS/MS data due to small or coeluting peaks.

[0246]To test the repeatability and the reproducibility of the system, the
NUTRILON® Pepti Plus sample was extracted twice and analyzed in
triplicate (in the beginning of the series, in the middle and at the
end). The data obtained from the various analyses on the distribution of
the carboxy-terminal amino acid residues were found to be in good
agreement.

[0247]The molar incidence of peptides carrying carboxy-terminal proline
residues in the various commercial products is provided in Table 3. The
molar incidence of such peptides is also related to the proline content
of the proteinaceous raw material used for preparing the hydrolysate. For
example casein and collagen have much higher proline contents than whey
or soy proteins. To take this aspect into account the molar fractions of
proline among the amino acids present in the protein basis used for each
commercial product has also been deduced using acid hydrolysis followed
by amino acid analysis using techniques as described in the Materials &
Methods section. Moreover raw material used can differ in their
susceptability to enzyme cleavage, for example because of the presence of
specific repeating amino acid sequences.

[0248]From the data presented in Table 3 it is clear that in the popular
whey hydrolysates the molar incidence of peptides carrying
carboxy-terminal proline residues is low. If we also take the proline
content of whey into account, we conclude that none of the commercial
whey based products contains a molar fraction of peptides carrying
carboxy-terminal proline residues which is higher than the molar fraction
of proline occurring in the protein basis. Typically the molar fraction
of peptides carrying a carboxy-terminal proline in these whey based
commercial products is 5% or lower.

[0249]Looking at the molar incidence of carboxy-terminal proline residues
in a casein based product like NUTRAMIGEN®, we see a substantial
higher level than can be found in the whey based products even if the
relatively high proline content of casein is taken into account. However,
comparing the NUTRAMIGEN® product on the one hand with the
beta-casein hydrolysate made by incubation with subtilisin and a
proline-specific endo-protease (see Example 1) shows the vast
compositional difference that can occur between an existing commercial
casein hydrolysate and a casein hydrolysate according to the invention.
Whereas the commercial product (i.e., NUTRAMIGEN®) exhibits a molar
incidence of peptides carrying a carboxy-terminal proline residue of 22%,
this figure for the casein hydrolysate according to Example 1 is 61%.

[0250]In this Example a commercial whey protein was incubated under
various conditions with a proline-specific endoprotease as produced by E.
coli. In the resulting hydrolysate the molar incidence of peptides
carrying a carboxy-terminal proline residues was determined.

[0251]A solution of PROTARMOR® 905 (Armor Proteins) in water (10% w/w)
was slowly heated up from 25° C. to 60° C. during 1 hr in
the presence of 2.5% (weight enzyme/weight substrate) DELVOLASE® at
pH 8.5. After 1 hr the solution was quickly heated to 80° C. and
immediately cooled down to 60° C. after which a new 2.5% dosage of
DELVOLASE® was added. The hydrolysis was allowed to continue for
another hour; then heated to 95° C. for 5 min and cooled again.
After adjustment of the pH to 7.4 the proline-specific endoprotease was
added in concentrations of 0, 87 and 170 units/gram of substrate (U/g in
Table 4; units according to the procedure described in World Journal of
Microbiology & Biotechnology, 11, 209-212, 1995) and hydrolysis was
allowed to proceed for another 3 hr at 45° C. At the end the
solution was kept at 95° C. for 5 min to inactivate the enzyme and
to pasteurise the solution. The hydrolysates as obtained were then
analyzed by LC/MS to determine the molar incidence of carboxy-terminal
proline residues in the peptides formed as described previously. The
results obtained are presented in Table 4.

[0252]From this Table, it appears that at 45° C. the molar
incidence of peptides carrying proline at their C-terminus increases with
the dose of the proline-specific endoprotease. Using the highest enzyme
dosages, up to 50% of the peptides obtained from this whey product could
be shown to carry a carboxy-terminal proline residue. When the incubation
is performed at 30° C., the molar incidence of peptides carrying a
carboxy-terminal proline residue can reach 52% with 87 units/gram
substrate and is hardly increased with higher doses of the enzyme. The
higher incidence reached with 87 U/g at 30° C. compared to
45° C. might be explained by a low thermostability of the E. coli
enzyme.

Example 8

Taste and Composition of Whey Hydrolysates Produced With or Without
Proline-Specific Endoprotease

[0253]In this Example a proline-specific endoprotease obtained from E.
coli was used in combination with subtilisin (DELVOLASE®) to produce
a whey hydrolysate of low bitterness. Using the data generated in Example
7 the dosage of the proline-specific endoprotease was chosen such that
only a marginal increase of peptides carrying a carboxy-terminal proline
residues could be expected. The hydrolysate formed with the
proline-specific endoprotease was compared with a similar hydrolysate
formed without a proline-specific endoprotease as well as a commercial,
low bitter whey hydrolysate. All three products were characterized in
terms of taste and their content of peptides carrying a carboxy-terminal
proline residue.

[0254]A solution of Protarmor® 905 (Armor Proteins) in water (10% w/w)
was slowly heated up from 25° C. to 60° C. during 1 hr in
the presence of 2.5% (weight enzyme/weight substrate) DELVOLASE® at
pH 8.5. After 1 hr the solution was quickly heated to 80° C. and
immediately cooled down to 60° C. after which a new 2.5% dosage of
DELVOLASE® was added. The hydrolysis was allowed to continue for
another hour; then heated to 95° C. for 5 min and cooled again.
After adjustment of the pH to 7.4 the proline-specific endoprotease was
added in a concentration of 50 units/gram of substrate. This was allowed
to continue for 3 hr at 45° C. According to the data obtained in
Example 7 these conditions lead to a marginal increase in peptides
carrying a carboxy-terminal proline residue only. At the end the solution
was kept at 95° C. for 5 min to inactivate the enzyme and to
pasteurise the solution. Then the solution was cooled down. The same
treatment was applied to another sample but without adding the
proline-specific endoprotease.

[0255]Sensorial analysis of the hydrolysates was carried out in so called
two-paired comparison tests. This type of test is used by the American
Society of Brewers Chemists (ASBC) to compare the bitterness of 2
different beers. If we accept a 5% risk of error in such a one-sided
test, the threshold value for having a statistical difference is 17 out
of 24 replies. In each test, the hydrolysates were tasted in 2.5% dry
matter concentrations and 1 ml portions of each solution were presented
in a disposable vial. Each assessor was asked to rate the bitterness
level without swallowing and to rinse the mouth with water afterwards.
All samples were coded and allotted at random among the assessors.

[0256]The first test was aimed at evaluating the benefit of the
combination of subtilisin and the proline-specific endoprotease versus
subtilisin alone. The second test was aimed at evaluating the bitterness
of the hydrolysate obtained with the combination of subtilisin and
proline-specific endoprotease versus a commercial, low bitter hydrolysate
(VITALARMOR® 800LB). To that end the VITALARMOR® 800LB was
diluted in the same buffer as used for the other hydrolysate to obtain a
comparable protein concentration.

[0257]Of the 24 persons participating in the first test, 17 rated the
sample obtained with the combination of subtilisin and proline-specific
endoprotease as less bitter than the sample obtained with subtilisin
alone. This result is statistically significant and confirms the
debittering activity of a proline-specific endoprotease, even if applied
at relatively low concentrations (cf. Example 7). Worthwhile to note is
that these "low"enzyme concentrations are several orders of magnitude
higher than the enzyme dosages applied in JP 05-015314 and for which a
debittering effect was claimed.

[0258]In the second paired sample comparison, 19 out of the 24
participants rated the sample treated with the combination of subtilisin
and proline-specific endoprotease as less bitter than the commercial
VITALARMOR® 800LB product. The latter observation is statistically
also significant and illustrates the economical value of the hydrolysates
and enzyme mixtures of the invention.

[0259]The hydrolysates obtained with or without the proline-specific
endoprotease were analyzed by LC/MS as described before. In the
hydrolysate obtained with the subtilisin alone, 41 peptides were
analyzed. It came out that none of these peptides carried a
carboxy-terminal proline residue despite the fact that 18 peptides were
shown to contain at least one proline residue.

[0260]In the hydrolysate obtained with the combination of subtilisin and
proline-specific endoprotease 31 peptides were analyzed and 6 were shown
to carry a carboxy-terminal proline residue. This observation, which is
in line with what could be expected on the basis of the results obtained
in Example 6, shows that as the result of the incubation with the
proline-specific endoprotease the molar incidence of peptides bearing a
carboxy-terminal proline residue was increased from 0 to 19%. As the
sensory analysis of the latter products has demonstrated a statistically
significant reduced bitterness, this experiment clearly links a slight
increase in the molar incidence of carboxy-terminal proline residues with
reduced bitterness.

[0261]Apart from decreasing the level of bitterness, this incubation with
a low level of proline-specific endoprotease could also be shown to
decrease the peptide length of the hydrolysate. In the hydrolysate
treated with DELVOLASE® alone, the LC/MS analysis revealed that
peptides vary in length from 4 to 14 amino-acids with an average length
of 7.5 amino-acids. In the hydrolysate treated with the combination of
DELVOLASE® and the proline-specific endoprotease, the peptide length
could be shown to vary from 4 to 12 amino-acids with an average length of
6.1 amino-acids. These reduced peptide lengths will not only improve the
yield of the hydrolysate production process, but also reduce the overall
allergenicity of the hydrolysate and minimise precipitation under acid
conditions.

Example 9

[0262]Cloning of Proline-Specific Endoprotease from Asperqillus niger

[0263]Forward and reverse oligonucleotide primers were developed using the
peptide sequences that were elucidated in Example 3. To reduce degeneracy
of the primers, inosine bases were introduced at several positions. This
increases the abundance of oligonucleotide primers in the pool that are
able to prime a PCR reaction, but the disadvantage is that the
specificity of the reaction decreases. Genomic DNA from A. niger G306
(deposited as CBS109712 with the CBS on Sep. 10, 2001) was isolated using
standard techniques and used as template in PCR reaction with the
oligonucleotide primers indicated in Table 5.

[0264]In the experiment all possible combinations of forward and reverse
primers were used to amplify the gene encoding the proline-specific
endoprotease from A. niger. Initial experiments were performed under
standard PCR conditions (denaturation at 94° C., annealing at
55° C. and extension at 72° C.). Surprisingly these
experiments did not yield any specific PCR product. Since a negative
result might also be due to impurities in the template DNA, we performed
control PCR reactions using PCR primers for several different but known
A. niger genes. In comparable reactions these latter genes could be
successfully amplified from A. niger G306 genomic DNA, showing that the
inability to amplify a fragment using the endo-Pro primers was not due to
impurities in the genomic DNA preparation.

[0265]Subsequently it was decided to decrease the stringency of the PCR
reaction, by decreasing the annealing temperature down to 45° C.
Consequently the specificity of the PCR was decreased and several bands
were amplified, although most of these bands were also detected in
control PCR reactions lacking one of the primers. Several of these PCR
products were cloned into the general cloning vector pCR2.1 (Invitrogen,
Groningen, Netherlands), and the DNA sequence of these fragments was
determined. Unfortunately none of the cloned fragments coded for the gene
encoding proline-specific endoprotease.

[0266]Additionally, many other adjustments to the PCR protocol were made
such as the use of a different polymerase, increasing primer- or
template-concentration, a touch-down PCR and introduction of a hot start,
but none of these protocols yielded a specific fragment of the gene
encoding the proline-specific endoprotease. To minimize the obvious risks
of this uncertain approach, it was decided to try another, less
well-known cloning procedure.

3'-RACE

[0267]Since none of our attempts to amplify the gene encoding the
proline-specific endoprotease from A. niger G306 genomic DNA were
successful, we decided to use a different approach in which RNA is used
as the template for cDNA synthesis. The approach of cloning an unknown
gene using 3'-RACE, 5'-RACE and amplification of the complete open
reading frame, has been described in WO 99/38956. The advantage of this
procedure, compared to the direct PCR procedure described above, is that
an additional priming site is introduced at the 3'-end of the cDNA, so
that only a single gene-specific oligonucleotide plus an universal primer
is required to amplify part of the coding sequence, instead of two
degenerate primers. Additionally, using cDNA as template circumvents
problems in amplification due to introns. The use of cDNA as template in
the amplification reaction also increases the concentration of the
template compared to amplification from genomic DNA.

[0268]According to this approach, A. niger G306 was grown in a medium
containing collagen as sole carbon source to induce the expression of the
gene encoding for proline-specific endoprotease. Medium composition is
described in the Materials & Methods section. Young mycelium was
harvested after 48 hr growth at 34° C., and used for the isolation
of total RNA. To this end, mycelium was harvested using filtration
through Miracloth filtration wrap and washed with ice cold sterile
demiwater. Mycelium (250 mg) was frozen immediately in liquid nitrogen
and ground to a fine white powder using mortar and pestle. The white
powder was transferred to a sterile 15 ml Greiner tube and total RNA was
isolated with the TRIZOL method exactly as described by the supplier
(Life Technologies, Paisley, UK).

[0269]The RNA preparation was used to synthesize cDNA from the anchor
primer of the 3'-RACE kit (AP; Life Technologies), extending cDNA from
the poly-A tail of mRNA. After RNase H treatment, cDNA was amplified by
PCR with the abridged universal amplification primer (AUAP; Life
Technologies) and the inosine substituted gene-specific forward primers
(SEQ ID NOS: 4, 7, 10 and 13) described above. Only with the first primer
SEQ ID NO: 4 plus AUAP could a specific amplification product of
˜1.4 kb be amplified from A. niger G306 RNA. With the other primers
only non-specific amplification at low stringency was obtained. This 1.4
kb cDNA fragment was cloned into pCR2.1 and the DNA sequence was
determined.

5'-RACE

[0270]From this sequence three gene-specific primers were designed for
further amplification of the 5'-part of the gene. All three primers,
5'-TTCAGTACT CCACCAGTACCTC-3' (SEQ ID NO: 16),
5'-TGGGAAAAGGTGCCCTTCTCC-3' (SEQ ID NO: 17), and
5'-GGATTATGATGGTCCAGCAGC-3' (SEQ ID NO: 18), were complementary and
reverse to the coding sequence of the gene coding for proline-specific
endoprotease.

[0271]Total RNA from A. niger G306 was used to synthesize cDNA with the
5'-RACE kit (Life Technologies). using primer
5'-TTCAGTACTCCACCAGTACCTC-3' (SEQ ID

[0272]NO: 16). After RNase treatment, cDNA was purified using the
GLASMAX® cartridge (Life Technologies). A poly-dC tail was added to
the cDNA using terminal transferase (TdT; Life Technologies). The cDNA
was amplified in a PCR reaction using the abridged anchor primer (AAP;
Life Technologies) and with the first nested primer
5'-TGGGAAAAGGTGCCCTTCTCC-3' (SEQ ID NO: 17). A second amplification
reaction using the AUAP primer (Life Technologies) and a second primer
5'-GGATTATGATGGTCCAGCAGC-3' (SEQ ID NO: 18) was required to obtain a
specific amplification product of ˜0.25 kb. This fragment was
purified via agarose gel electrophoresis and cloned into pCR2.1 and the
DNA sequence was determined. This showed that this fragment contains the
5'-part of the gene coding for the proline-specific endoprotease.

Characterization of the Gene

[0273]Combining the overlapping sequences of the 3'-RACE and the 5'-RACE
results in the complete coding sequence of the gene encoding the
proline-specific endoprotease. SEQ ID NO: 1 shows the entire sequence of
the open reading frame of this gene. The deduced protein sequence of 526
amino acids is depicted in SEQ ID NO: 2. Peptide ATTGEAYFE (SEQ ID NO: 3)
appeared to be completely correct. Peptide DGAPEGTST (SEQ ID NO: 9) is
also correct but is encoded by genomic DNA that is interrupted by an
intron (see SEQ ID NO: 15 and example 11 for the cloning and sequence of
genomic DNA of Aspergillus niger CBS513.88). The other two peptides
incorporate errors due to the LC/MS/MS approach which has been used for
their characterization (see Example 3). Despite these uncertainties we
successfully selected and identified the desired genetic information
encoding the proline-specific endoprotease from Aspergillus for the first
time.

[0274]The novelty of the proline-specific endoprotease from Aspergillus
was confirmed by BLAST searches to well-known databases such as
SwissProt, PIR and trEMBL. No strong identity of this protein with any
other protein can be detected when compared to the protein sequence
databases.

[0275]The entire open reading frame of the gene encoding proline-specific
endoprotease was PCR amplified from cDNA of A. niger G306 using the
primers 5'-ATGCGTGCCTTCTCCGCTGTC-3' (see bases 1 to 21 of SEQ ID NO: 1)
and the AUAP primer (Life Technologies). The obtained PCR fragment was
cloned into the cloning vector pCR2.1 (Invitrogen). The resulting plasmid
was digested with EcoRI and the fragment containing the endo-Pro gene was
cloned into the EcoRI site of expression vector pGBFIN-11 (WO 99/32617).
The resulting clones were checked by restriction with XhoI, which yields
a fragment of ˜0.65 kb when the fragment is inserted in the correct
orientation. The resulting plasmid is shown in FIG. 1 and was named
pGBFIN11-EPO.

[0276]A. niger CBS513.88 was used as host for the over-expression of the
gene encoding the proline-specific endoprotease. Therefore, the
expression vector pGBFIN11-EPO was linearized by digestion with NotI,
which removes all E. coli derived sequences from the expression vector.
The digested DNA was purified using phenol:chloroform:isoamylalcohol
(24:23:1) extraction and precipitation with ethanol. The A. niger
transformation procedure is extensively described in. WO 98/46772. It is
also described how to select for transformants on agar plates containing
acetamide, and to select targeted multicopy integrants. Preferably, A.
niger transformants containing multiple copies of the expression cassette
are selected for further generation of sample material.

Cultivation and Isolation of Protease

[0277]An A. niger strain containing multiple copies of the expression
cassette was used for chromatographic generation of sample material by
cultivation of the strain in shake flask cultures. A useful method for
cultivation of A. niger strains and separation of the mycelium from the
culture broth is described in WO 98/46772. The culture broth obtained was
analyzed on SDS-PAGE which is depicted in FIG. 2 Subsequently, the
culture broth was used for chromatographic purification of the protease
to remove any contaminating endo- and exoproteolytic activities. To that
end the fermentation broth was first centrifuged to remove the bulk of
the fungal mass and the supernatant was then passed through a number of
filters with decreasing pore sizes to remove all cell fragments. Finally,
the ultrafiltrate obtained was diluted ten times in 20 mmol/liter sodium
acetate pH 5.1 and applied on a Q-Sepharose FF column. Proteins were
eluted in a gradient from 0 to 0.4 moles/liter NaCl in 20 mmol/liter
sodium acetate pH 5.1. Peak fractions displaying activity towards the
cleavage of Z-Gly-Pro-pNA (Bachem, Switzerland) were collected and
pooled, according to the protocol described in World Journal of
Microbiology & Biotechnology 11, 209-212 (1995), but under slightly
modified assay conditions. Taking the acid pH optimum of the A. niger
derived proline-specific endoprotease into account, the enzyme assay was
carried out at pH 5 in a citrate/phosphate buffer at 37° C.
Pooling of the active fractions followed by concentration finally yielded
a preparation which showed only a single band on SDS-PAGE and one peak on
HP-SEC. Further analysis by hydrophobic interaction chromatography
confirmed the purity of the enzyme preparation obtained.

[0278]Furthermore, the purified proline-specific endoprotease was used for
the determination of the amino-terminus of the mature protein, by Edman
degradation. The amino-terminus of the mature proline-specific
endoprotease starts at position 42 in SEQ ID NO: 2 and SEQ ID NO: 17.

Example 11

[0279]Screening of Fungal Species Other than A. niger for Presence of Gene
Encoding Proline-Specific Endoprotease

[0281]When cultures were sufficiently grown, mycelial mass was harvested
by filtration through Miracloth filter, washed with 10 mM KPi buffer (pH
7.0) and dried between filterpaper. Mycelium was ground under liquid
nitrogen with a mortar and pestle, until a fine white powder was
obtained. Subsequently, chromosomal DNA was isolated using the PureGene
kit (Gentra Systems, Minneapolis USA) according to instructions by the
supplier.

[0282]Saccharomyces cerevisiae ATCC20785 was used as negative control in
the experiment and cultivated in YePD at 30° C. and shaken at 220
rpm. For preparation of a Southern blot, chromosomal DNA of all species
was digested with XhoI and restriction fragments were separated by
agarose gelelectrophoresis on a 0.8% agarose gel in TAE buffer. After
separation, DNA fragments were blotted to nitrocellulose (0.2 μm,
Schleicher & Schuell) membranes by conventional procedure (Sambrook et
al., Molecular Cloning, ISBN 0-87969-309-6, 1982), and the blot was baked
for 2 hr at 80° C.

[0283]The probe for hybridization was synthesized with PCR on pGBFIN11-EPO
as template using primers 5'-ATGCGTGCCTTCTCCGCTGTC-3' (see bases 1 to 21
of SEQ ID NO: 1) and the AUAP primer. About 30 nanograms of the cDNA
fragment was labeled with 32P-alpha-dATP (Amersham, England) with
the RadPrime DNA labeling system (Life Technologies) according to the
suppliers instructions. After labeling unincorporated dNTP's were removed
by purifying the probe fragment over a Sephadex G-50 column according to
the spun-column procedure (Sambrook et al., 1982).

[0284]Prior to adding to the hybridization mixture, the purified probe was
denatured by incubation in boiling water for 5 min followed by rapid
cooling in ice, and used immediately.

[0285]Prehybridization of the blots was in 50 ml 6×SSC, 0.5% SDS,
5× Denhardt, 0.1 mg/ml Herring sperm DNA (Life Technologies) for 1
hr at 50° C. under continuous agitation. After addition of the
probe to the prehybridization solution, hybridization was performed for
16 hr at 50° C. The blots were washed twice with 200 ml
6×SSC, 0.1% SDS for 30 min at ambient temperature, and once with
200 ml 6×SSC, 0.1% SDS for 30 min at 50° C., to remove
aspecific hybridization to the blot. X-Omat AR (Kodak) films were used to
visualize the hybridization.

[0286]The results of this experiment are depicted in Table 6. A. niger and
A. carbonarius strains give strong hybridization with the probe. Also
other Aspergillus strains like A. sojae, A. ochraceus and A. acculeatis
give hybridization with the probe. Apparently the gene encoding the
proline-specific endoprotease is well conserved within the Aspergillus
genus. Surprisingly, also fungi that are more distant from Aspergillus,
like Phialophora mustea, Rhizomucor miehei, Alternaria alternata,
Talaromyces emersonii, and Trichoderma reesii give good hybridization to
the cDNA of the proline-specific endoprotease. Saccharomyces cerevisiae
which was included as negative control, as well as a few other species do
not show any hybridization with the cDNA from A. niger (see Table 6).
This result shows that the gene encoding the proline-specific
endoprotease is conserved in many fungal species, and a person skilled in
the art will understand that the genes from these species can be isolated
using the heterologous hybridization shown here as detection method.

[0287]To illustrate this, the cDNA fragment of Aspergillus niger G306,
used in this example, was used as probe for the screening of a genomic
DNA library of Aspergillus niger CBS513.88. A person skilled in the art
will have knowledge to generate a genomic DNA library, and to screen such
a library with a labelled DNA probe. This procedure has also been
described extensively in literature (Sambrook et al., Molecular Cloning,
CSHL Press, 1989). Positive clones in the screening were purified and the
DNA was sequenced. Aspergillus niger CBS513.88 genomic DNA coding for the
proline-specific endoprotease is represented in SEQ ID NO: 15. This
example illustrates that it is possible to isolate the gene coding for
the proline-specific endoprotease from other species and strains using
hybridization to the cDNA of this gene from Aspergillus niger G306.

[0289]Enzyme Mixture Obtained from Asperqillus oryzae FS 1-32 and its
Effects in Hhydrolysis of Soy Protein

[0290]Japanese patent JP 05-015314 discloses a crude enzyme preparation
obtained from Aspergillus oryzae FS 1-32 containing major quantities of a
non-specified endoproteolytic activity and minor quantities of a
proline-specific endoprotease. This crude preparation further contains a
significant carboxypeptidase activity. Upon incubation of soy bean
protein with this crude enzyme preparation, a soy bean protein
hydrolysate is obtained that is claimed to be significantly less bitter
than a soy bean hydrolysate that can be obtained with other protease
preparations. The explanation given in JP 05-015314 for this beneficial
debittering effect is that other protease preparations lack the presence
of a proline-specific endoprotease in combination with a
carboxypeptidase. JP 05-015314 suggests that the basis for the
debittering effect is the removal of the proline residues that are
exposed by the activity of the proline-specific endoprotease and
subsequently removed by the carboxypeptidase.

[0291]Example 4 of the present application describes the effects on soy
protein of a mixture of commercial enzymes resembling the proteolytic
activity profile of strain A. oryzae FS 1-32. One of the conclusions of
this experimental work is that the incorporation of a proline-specific
endoproteolytic activity in levels as recorded with strain FS 1-32
doesnot lead to an appreciable increase in soy peptides carrying a
carboxy-terminal proline residue. As this conclusion has important
implications regarding the non-bitter protein hydrolysates described in
the present application, we decided to repeat the experiment but using
the enzyme mixture as obtained from A. oryzae FS 1-32 and under
conditions as described in JP 05-015314.

[0292]Aspergillus oryzae FS 1-32 (as obtained from depot 12193 of the
Micr. Ind Lab in Japan) was plated on malt extract agar plates, incubated
for four days at 35° C. and then stored for one day at 4°
C. Spores from these plates were used to inoculate the inoculation medium
containing 20 grams/kg of dextrose, 15 grams/kg of defatted soy flour, 5
grams/kg of low salt yeast extract, 1 gram/kg of KH2PO4 and 0.2 grams/kg
of antifoam. After dissolution in demineralised water, the pH of the
medium was adjusted with sulphuric acid to 5.5 and then divided in
portions of 20 ml over 100 ml shakeflasks with baffles. Shakeflasks with
medium were sterilized for 30 min at 121° C. and inoculated after
cooling down. After two days in a shake incubator at 32° C., 1 ml
was used to inoculate another 100 ml inoculation medium. After another
day in the shake incubator at 32° C. this culture was used to
inoculate the culture medium. Because JP 05-01314 did not provide
information regarding the fermentation procedures used, the fermentation
protocol and medium as provided in EP 0522428 was used.

[0294]KH2PO4 26.6 grams/liter. Because the recommended tannic
acid (to stimulate the formation of the proline-specific endoprotease)
was not specified in EP 0522428, two kinds of tannic acids, i.e., BREWTAN
C and TANAL W2 (both from Omnichem (Wetteren, Belgium) were used. Finally
the pH value of the culture medium was adjusted with phosphoric acid
(20%) to 4.5 and then divided in portions of 100 ml in 500 ml shake
flasks with baffles. Flasks were sterilized for 30 min at 121° C.

[0295]After inoculation with 1 ml of the pre-grown inoculation medium, the
cultures were incubated for 2 and 4 days at 32° C., 250 rpm. To
remove the biomass, the culture broths were filtered over a Whatman glass
microfibre filter (cat no 1820090) which were then stored at -20°
C. Part of this frozen material was lyophilized and used for activity
measurements as well as incubations with soy protein.

[0296]The activities of the prolyl-endopeptidase, carboxypeptidase and
endoprotease in the lyophilized materials were measured exactly as
described in JP 05-015314. The samples that had been fermented for 2 days
showed appreciably higher enzyme activity levels then the samples that
had been fermented for the recommended 4 days so that it was decided to
use these 2 days samples for the final incubation with soy protein.
Enzyme activity data of those samples showing the highest
prolyl-endopeptidase activities are shown hereunder.

[0297]The propyl-endopeptidase and the carboxypeptidase activities
measured in the samples 1, 3 and 4 are comparable with the figures
provided in JP 05-015314. However, the endoprotease activities measured
in these samples turned out to be about 200 times lower than indicated in
JP 05-01314. In view of the endoproteolytic activities reported in
various industrial enzyme preparations (see Example 4), the extremely
high endoproteolytic activities obtained with A. oryzae FS 1-32 and
specified in JP 05-015314 are probably unrealistic.

[0298]In an attempt to copy Example 2 of JP 05-015314 as precisely as
possible, the following experiment was carried out. Ten grams of soy
protein SOYAMIN® 90HV (Lucas Meyer, Hamburg, Germany) were suspended
in 100 ml demineralized water and the pH was adjusted with 4N NaOH to
8.5. Then 0.5 g DELVOLASE® (DSM Food Specialities, Seclin, France)
was added (instead of PROTIN AY® from Daiwa Kasei; both DELVOLASE®
and PROTIN AY® are Bacillus-derived alkaline endoproteases) and the
protein solution was incubated for 2 hr at 60° C. (in JP 05-015314
the incubation time and temperature with PROTIN AY® are not
specified). Finally the DELVOLASE® was inactivated by heating the
solution for 10 min at 92° C.

[0299]The resulting protein hydrolysate was then incubated with the enzyme
samples 1, 3 and 4 according to the protocol described in JP 05-015314
but standardized according to the desired carboxypeptidase activity (0.01
unit per gram substrate). The implication was that per gram of soy
isolate 2.0 mg of lyophilized enzyme sample 1 had to be added, 2.7 mg of
lyophilized enzyme sample 3 and 1.3 mg of lyophilized enzyme sample 4.
The resulting endoprotease and prolyl endoprotease activities are
presented in Table 8.

[0300]After incubation for 5 hr at pH 5 and 50° C., the samples
were centrifuged and the supernatants were kept frozen until LC/MS
analysis.

[0301]LC/MS analysis was carried out as specified in the Materials &
Methods section.

[0302]In this experiment the protein data bank consisted of soy proteins
only. The frequency of carboxy-terminal proline residues detected in the
peptides obtained are specified underneath.

[0303]From the data obtained it is obvious that the incubation of soy
protein with the crude enzyme preparation obtained from Aspergillus
oryzae FS 1-32 doesnot result in a significant increase of the molar
fraction of peptides carrying a carboxy-terminal proline residue. So the
debittering effect described in JP 05-015314 cannot be attributed to a
high incidence of such peptides in the final hydrolysate.

[0305]Proline-specific endoprotease from A. niger G306 was overexpressed
and chromatographically purified (see Example 10) and subsequently used
to produce a non-bitter casein hydrolysate. To that end we added to 100
mL of a solution of sodium caseinate (MIPRODAN® 30) containing 60
grams per liter, 100 mg of thermolysin (THERMOASE®). Incubation at pH
6.7 and 85° C. resulted in an immediate flocculation and
precipitation of caseinaceous protein. Incubation for two hours finally
resulted in a clarified solution still containing some precipitate. Then
the pH of the solution was adjusted to pH 5.0 and the THERMOASE® was
inactivated by heating for 45 min at 95° C. After cooling down,
the solution was tasted and observed to be very bitter. In this stage the
DH (Degree of Hydrolysis; established using the TNBS method) of the
caseinate solution was approx 35%. Analysis of 64 peptides by LC/MS/MS
using a databank for bovine caseinates indicated a molar incidence of
peptides carrying a carboxy-terminal proline residue of 14%.

[0306]Then 3 units of the chromatographically purified proline-specific
endoprotease from A. niger were added to 25 ml of the hydrolysate. After
incubation for 20 hr at 50° C., another enzyme inactivation cycle
was carried out by heating the solution for 30 min at 90° C. After
cooling to room temperature the solution was decanted and the clear
supernatant was adjusted to a pH value to 4.0; the caseinate hydrolysate
was found to remain completely dissolved and clear. Tasting demonstrated
the absence of any bitterness or off-flavors. The DH of this final
hydrolysate using the TNBS method was approx 50%; LC/MS/MS analysis of 64
peptides showed that the molar incidence of peptides carrying a
carboxy-terminal proline residue was increased to 45%. This 45% is almost
4 times higher than the molar fraction of proline occurring in the
MIPRODAN® substrate.